Compositions containing non-natural amino acid linked dolastatin derivatives, methods therewith, and uses thereof - Patents.com

Abstract

Provided are unnatural amino acids, dolastatin analogs that include at least one unnatural amino acid, and methods for producing such unnatural amino acids and polypeptides. [Solution] Dolastatin analogs have at least one oxime group, carbonyl group, dicarbonyl group, and / or hydroxylamine group. Also disclosed are unnatural amino acid dolastatin analogs that are further post-translationally modified, methods for effecting such modifications, and methods for purifying such dolastatin analogs. Modified dolastatin analogs typically have at least one oxime group, carbonyl group, dicarbonyl group, and / or hydroxylamine group. Additionally, methods for using such unnatural amino acid dolastatin analogs and modified unnatural amino acid dolastatin analogs, including therapeutic, diagnostic, and other biotechnological uses, are disclosed.

Description

Detailed Description of the Invention

[0001] [Cross reference] This application claims priority from U.S. Provisional Application No. 61 / 491,146, entitled "Compositions Containing Non-Natural Amino Acid-Linked Dolastatin Derivatives, Methods Thereof, and Uses Thereof," filed May 27, 2011, which is hereby incorporated by reference in its entirety.

[0002] BACKGROUND OF THE INVENTION The ability to incorporate amino acids that are not genetically encoded (i.e., "unnatural amino acids") into proteins allows for the introduction of chemical functional groups that may provide useful alternatives to naturally occurring functional groups (e.g., -NH2 at the epsilon position of lysine, the sulfhydryl -SH of cysteine, and the imino group of histidine, etc.). Some chemical functional groups are inert toward functional groups found on the 20 common genetically encoded amino acids, but are known to react efficiently and precisely to form stable bonds with functional groups that can be incorporated into unnatural amino acids.

[0003] Here we provide a method for selectively introducing chemical functional groups that are not present in proteins, are chemically inert toward all functional groups present in the 20 common genetically encoded amino acids, and can be used to react efficiently and selectively with reagents containing the specific functional group to form stable covalent bonds.

[0004] Summary of the Invention Disclosed herein are toxic moieties with one or more linkers, toxic groups attached to unnatural amino acids, and methods for producing such unnatural amino acids and polypeptides.

[0005] In some embodiments of the present invention, compounds are described according to formula (I), or salts thereof:

[0006] [ka]

[0007] (In the formula, Z has the following structure:

[0008] [ka]

[0009] R5 is H, COR8, alkyl having 1 to 6 carbon atoms, or thiazole; R8 is OH or -NH-(alkylene-O) n -NH2, R6 is OH or H; Ar is phenyl or pyridine; R7 is alkyl having 1 to 6 carbon atoms or hydrogen; Y is selected from the group consisting of hydroxylamine, methyl, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, azide, ketoamine, ketoalkyne, alkyne, cycloalkyne, and enedione; L is -alkylene-, -alkylene-C(O)-, -(alkylene-O) n -Alkylene-, -(alkylene-O) n -Alkylene-C(O)-, -(Alkylene-O) n -(CH2) n’ -NHC(O)-(CH2) n’’ -C(Me)2-SS-(CH2) n’’’ -NHC(O)-(alkylene-O) n’’’’ -Alkylene, -(alkylene-O) n -Alkylene-W-, -Alkylene-C(O)-W-, -(Alkylene-O) n -alkylene-U-alkylene-C(O)-, and -(alkylene-O) n -alkylene-U-alkylene; W has the following structure:

[0010] [ka]

[0011] U has the structure:

[0012] [ka]

[0013] Or, L is absent, Y is methyl, R5 is COR8, and R8 is -NH(alkylene-O) n -NH2, and n, n', n'', n''', and n'''' are each independently an integer of 1 or greater. In some embodiments, R5 is thiazole. In other embodiments, R6 is H. In some embodiments, Ar is phenyl. In further or additional embodiments, R7 is methyl. In some embodiments, n is an integer from 0 to 20, 0 to 10, or 0 to 5.

[0014] In some embodiments, compounds according to formula (II) are described.

[0015] [ka]

[0016] In certain embodiments, L is -(alkylene-O) n In certain embodiments, each alkylene is -CH2CH2-, n=3, and R7 is methyl. In other embodiments, L is -alkylene-. In certain embodiments, each alkylene is -CH2CH2-, and R7 is methyl or hydrogen. In certain embodiments, L is -(alkylene-O) n In some embodiments, each alkylene is -CHCH-, n=4, and R is methyl. In further or additional embodiments, L is -(alkylene-O).n -(CH2) n’ -NHC(O)-(CH2) n’’ -C(Me)2-SS-(CH2) n’’’ -NHC(O)-(alkylene-O) n’’’’ In some embodiments, each alkylene is -CHCH-, n is 1, n' is 2, n'' is 1, n''' is 2, n'''' is 4, and R is methyl.

[0017] In some embodiments, Y is azide. In other embodiments, Y is cyclooctyne. In particular embodiments, the cyclooctyne has the structure:

[0018] [ka]

[0019] (In the formula, Each R 19 are independently selected from the group consisting of alkyl having 1 to 6 carbon atoms, alkoxy having 1 to 6 carbon atoms, ester, ether, thioether, aminoalkyl, halogen, alkyl ester, aryl ester, amide, aryl amide, alkyl halide, alkylamine, alkylsulfonic acid, alkylnitro, thioester, sulfonyl ester, halosulfonyl, nitrile, alkylnitrile, and nitro; q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.) In some embodiments of the present invention, compounds are described according to formula (II), (III), (IV), (V), or (VI), or salts thereof:

[0020] [ka] JPEG0007638942000008.jpg115169

[0021] wherein Z has the structure:

[0022] [ka]

[0023] R5 is H, COR8, alkyl having 1 to 6 carbon atoms, or thiazole; R8 is OH; Ar is phenyl or pyridine; R7 is alkyl having 1 to 6 carbon atoms or hydrogen; Y and V are each selected from the group consisting of hydroxylamine, methyl, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, azide, ketoamine, ketoalkyne, alkyne, cycloalkyne, and enedione; L1, L2, L3, and L4 each independently represent a bond, -alkylene-, or -(alkylene-O) n -Alkylene-J-, -Alkylene'-J-(Alkylene-O) n -Alkylene-, -J-(Alkylene-O) n -Alkylene-, -(Alkylene-O) n -Alkylene-J-(Alkylene-O) n '-Alkylene-J'-, -(Alkylene-O) n -Alkylene-J-alkylene'-, -W-, -Alkylene-W-, Alkylene'-J-(Alkylene-NMe) n -Alkylene-W-, -J-(Alkylene-NMe) n a linker selected from the group consisting of -alkylene-W-, -J-alkylene-NMe-alkylene'-NMe-alkylene''-W-, and -alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene''''-W-; W has the following structure:

[0024] [ka]

[0025] Each J and J' independently has the structure:

[0026] [ka]

[0027] Each n and n' is independently an integer of 1 or greater.

[0028] In one embodiment, a compound comprising formula (VII) is described.

[0029] [ka]

[0030] In certain embodiments, L1 is -(alkylene-O) n -alkylene-J-, and L2 is -alkylene'-J'-(alkylene-O) n '-Alkylene-, L3 is -J''-(Alkylene-O) n ''-alkylene-, alkylene is -CH2CH2-, alkylene' is -(CH2)4-, n is 1, n' and n'' are 3; J has the following structure:

[0031] [ka]

[0032] J' and J'' have the following structure:

[0033] [ka]

[0034] R7 is methyl. In another embodiment, L1 is -J-(alkylene-O) n -alkylene-, and L2 is -(alkylene-O) n' -alkylene-J'-alkylene'-, and L3 is -(alkylene-O) n'' -alkylene-J″-, where alkylene is —CH2CH2—, alkylene′ is —(CH2)4—, n is 1, n′ and n″ are 4, and J, J′, and J″ have the following structure:

[0035] [ka]

[0036] In some embodiments, Y is azide. In other embodiments, Y is cyclooctyne. In particular embodiments, cyclooctyne has the structure:

[0037] [ka]

[0038] (In the ceremony Each R 19 are independently selected from the group consisting of alkyl having 1 to 6 carbon atoms, alkoxy having 1 to 6 carbon atoms, ester, ether, thioether, aminoalkyl, halogen, alkyl ester, aryl ester, amide, aryl amide, alkyl halide, alkylamine, alkylsulfonic acid, alkylnitro, thioester, sulfonyl ester, halosulfonyl, nitrile, alkylnitrile, and nitro; q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.) In one embodiment of the present invention, compounds are described according to formula (VIII) or (IX), or an active metabolite, pharmaceutically acceptable prodrug, or solvate thereof:

[0039] [ka]

[0040] (In the formula, A is optionally, if present, lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (where k is 1, 2, or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, at least one amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, at least one amino acid, a polypeptide, or a polynucleotide; R3 and R4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4, or two R3 groups, may optionally form a cycloalkyl or heterocycloalkyl; Z has the following structure:

[0041] [ka]

[0042] R5 is H, COR8, alkyl having 1 to 6 carbon atoms, or thiazole; R8 is OH; R6 is OH or H; Ar is phenyl or pyridine; R7 is alkyl having 1 to 6 carbon atoms or hydrogen; L is -alkylene-, -alkylene-C(O)-, -(alkylene-O) n -Alkylene-, -(alkylene-O) n -Alkylene-C(O)-, -(Alkylene-O) n -(CH2) n’ -NHC(O)-(CH2) n’’ -C(Me)2-SS-(CH2) n’’’ -NHC(O)-(alkylene-O) n’’’’ -Alkylene, -(alkylene-O) n -Alkylene-W-, -Alkylene-C(O)-W-, -(Alkylene-O) n -Alkylene-UC(O)- and -(Alkylene-O) n -alkylene-U-alkylene; W has the following structure:

[0043] [ka]

[0044] U has the structure:

[0045] [ka]

[0046] n, n', n'', n''', and n'''' are each independently an integer of 1 or greater. In some embodiments, R1 is a polypeptide. In certain embodiments, the polypeptide is an antibody. In certain embodiments, the antibody is Herceptin. In other embodiments, R2 is a polypeptide. In certain embodiments, the polypeptide is an antibody. In certain embodiments, the antibody is Herceptin.

[0047] In one embodiment of the present invention, a compound or salt thereof is described according to formula (X), (XI), (XII) or (XIII):

[0048] [ka] JPEG0007638942000022.jpg169169

[0049] (In the formula, A is optionally, if present, lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (where k is 1, 2, or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, at least one amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, at least one amino acid, a polypeptide, or a polynucleotide; R3 and R4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4, or two R3 groups, may optionally form a cycloalkyl or heterocycloalkyl; Z has the following structure:

[0050] [ka]

[0051] R5 is H, COR8, alkyl having 1 to 6 carbon atoms, or thiazole; R8 is OH; R6 is OH or H; Ar is phenyl or pyridine; R7 is alkyl having 1 to 6 carbon atoms or hydrogen; L1, L2, L3, and L4 each independently represent a bond, alkylene, -(alkylene-O) n -Alkylene-J-, -Alkylene'-J-(Alkylene-O) n -Alkylene-, -J-(Alkylene-O) n -Alkylene-, -(Alkylene-O) n -Alkylene-J-(Alkylene-O) n '-Alkylene-J'-, -(Alkylene-O) n -Alkylene-J-alkylene'-, -W-, -Alkylene-W-, Alkylene'-J-(Alkylene-NMe) n -Alkylene-W-, -J-(Alkylene-NMe) na linker selected from the group consisting of -alkylene-W-, -J-alkylene-NMe-alkylene'-NMe-alkylene''-W-, and -alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene''''-W-; W has the following structure:

[0052] [ka]

[0053] Each J and J' independently has the structure:

[0054] [ka]

[0055] Each n and n' is independently an integer of 1 or greater.

[0056] In some embodiments, R1 is a polypeptide. In certain embodiments, the polypeptide is an antibody. In certain embodiments, the antibody is Herceptin. In other embodiments, R2 is a polypeptide. In certain embodiments, the polypeptide is an antibody. In certain embodiments, the antibody is Herceptin.

[0057] In one embodiment, there is provided a method for derivatizing a dolastatin analog of formula (I), (III), (IV), (V), or (VI), comprising: The method includes contacting the dolastatin analog with a reagent of formula (XXXVII): Formula (I), (III), (IV), (V), or (VI) corresponds to:

[0058] [ka] JPEG0007638942000027.jpg185169

[0059] (In the formula, Z has the following structure:

[0060] [ka]

[0061] R5 is H, COR8, alkyl having 1 to 6 carbon atoms, or thiazole; R8 is OH or -NH-(alkylene-O) n -NH2, R6 is OH or H; Ar is phenyl or pyridine; R7 is alkyl having 1 to 6 carbon atoms or hydrogen; Y is NH2-O- or methyl; L, L1, L2, L3, and L4 each independently represent a bond, -alkylene-, -alkylene-C(O)-, or -(alkylene-O) n -Alkylene-, -(alkylene-O) n -Alkylene-C(O)-, -(Alkylene-O) n -(CH2) n’ -NHC(O)-(CH2) n’’ -C(Me)2-SS-(CH2) n’’’ -NHC(O)-(alkylene-O) n’’’’ -Alkylene, -(alkylene-O) n -Alkylene-W-, -Alkylene-C(O)-W-, -(Alkylene-O) n -Alkylene-J-, -Alkylene'-J-(Alkylene-O) n -Alkylene-, -(Alkylene-O) n -Alkylene-J-alkylene'-, -J-(alkylene-O) n -Alkylene-, -(Alkylene-O) n -Alkylene-J-(Alkylene-O) n '-Alkylene-J'-, -W-, -Alkylene-W-, Alkylene'-J-(Alkylene-NMe)n -Alkylene-W-, and -J-(Alkylene-NMe) n -Alkylene-W-, -(Alkylene-O) n -alkylene-U-alkylene; -J-alkylene-NMe-alkylene'-NMe-alkylene''-W-, and -alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene'''-W-; W has the following structure:

[0062] [ka]

[0063] U has the structure:

[0064] [ka]

[0065] Each J and J' independently has the structure:

[0066] [ka]

[0067] Or, L is absent, Y is methyl, R5 is COR8, and R8 is -NH(alkylene-O) n -NH2, and n, n', n'', n''', and n'''' are each independently an integer of 1 or greater. Formula (XXXVII) corresponds to the following, methods of which are described herein:

[0068] [ka]

[0069] (In the formula, A is optionally lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, if present; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(where k is 1, 2, or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; and each R' is independently H, alkyl, or substituted alkyl; K is

[0070] [ka]

[0071] and R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, at least one amino acid, or a polynucleotide; R2 is OH, an ester protecting group, a resin, at least one amino acid, or a polynucleotide; R3 and R4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4, or two R3 groups, may optionally form a cycloalkyl or heterocycloalkyl. In some embodiments, the derivatized dolastatin analog is an amino acid containing at least one oxime having the structure of formula (VIII), (IX), (X), (XI), (XII), or (XIII):

[0072] [ka] JPEG0007638942000035.jpg176169 JPEG0007638942000036.jpg169169

[0073] In certain embodiments, the dolastatin analog is contacted with the reagent of Formula (XXXVII) in aqueous solution under mildly acidic conditions.

[0074] In certain embodiments of the present invention, compounds are described according to formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX).

[0075] [ka] JPEG0007638942000038.jpg224169 JPEG0007638942000039.jpg103169

[0076] (In the formula, Z has the following structure:

[0077] [ka]

[0078] R5 is H, CO2H, alkyl having 1 to 6 carbon atoms, or thiazole; R6 is OH or H; Ar is phenyl or pyridine; R1 is H, an amino protecting group, a resin, at least one amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, at least one amino acid, a polypeptide, or a polynucleotide; R4 is H, halogen, lower alkyl, or substituted lower alkyl; R7 is alkyl having 1 to 6 carbon atoms or hydrogen; L, L1, L2, L3, and L4 each independently represent a bond, -alkylene-, -alkylene-C(O)-, -alkylene-J-, or -(alkylene-O) n -Alkylene-, -(alkylene-O) n -Alkylene-C(O)-, -(Alkylene-O) n -J-, -(alkylene-O) n -J-alkylene, -(alkylene-O) n -(CH2) n’ -NHC(O)-(CH2) n’’ -C(Me)2-SS-(CH2) n’’’ -NHC(O)-(alkylene-O) n’’’’ -Alkylene, -(alkylene-O) n -Alkylene-W-, -Alkylene-C(O)-W-, -(Alkylene-O) n -Alkylene-J-, -Alkylene'-J-(Alkylene-O) n -Alkylene-, -(alkylene-O) n-Alkylene-J-alkylene'-, -J-(alkylene-O) n -Alkylene-, -(alkylene-O) n -Alkylene-J-(Alkylene-O) n '-Alkylene-J'-, -W-, -Alkylene-W-, Alkylene'-J- (Alkylene-NMe) n -Alkylene-W-, -J-(Alkylene-NMe) n -Alkylene-W-, -(Alkylene-O) n -Alkylene-UC(O)-, -(Alkylene-O) n -alkylene-U-alkylene; -J-alkylene-NMe-alkylene'-NMe-alkylene''-W-, and -alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene'''-W-; W has the following structure:

[0079] [ka]

[0080] U has the structure:

[0081] [ka]

[0082] Each J and J' independently has the structure:

[0083] [ka]

[0084] n and n' are each independently an integer of 1 or greater; Each R 16 are independently selected from the group consisting of hydrogen, halogen, alkyl, NO, CN, and substituted alkyl. In some embodiments, R1 is a polypeptide. In certain embodiments, the polypeptide is an antibody. In certain embodiments, the antibody is Herceptin. In other embodiments, R2 is a polypeptide. In certain embodiments, the polypeptide is an antibody. In certain embodiments, the antibody is Herceptin.

[0085] In some embodiments of the present invention, compounds are described according to formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI).

[0086] [ka] JPEG0007638942000045.jpg195169 JPEG0007638942000046.jpg93169

[0087] (In the formula, Z has the following structure:

[0088] [ka]

[0089] R5 is H, CO2H, alkyl having 1 to 6 carbon atoms, or thiazole; R6 is OH or H; Ar is phenyl or pyridine; R1 is H, an amino protecting group, a resin, at least one amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, at least one amino acid, a polypeptide, or a polynucleotide; R4 is H, halogen, lower alkyl, or substituted lower alkyl; R7 is alkyl having 1 to 6 carbon atoms or hydrogen; L, L1, L2, L3, and L4 each independently represent a bond, -alkylene-, -alkylene-C(O)-, -alkylene-J-, or -(alkylene-O) n -Alkylene-, -(alkylene-O) n -Alkylene-C(O)-, -(Alkylene-O) n -J-, -(alkylene-O) n -J-alkylene, -(alkylene-O) n -(CH2) n’ -NHC(O)-(CH2) n’’ -C(Me)2-SS-(CH2) n’’’ -NHC(O)-(alkylene-O) n’’’’ -Alkylene, -(alkylene-O) n -Alkylene-W-, -Alkylene-C(O)-W-, -(Alkylene-O) n -Alkylene-J-, -Alkylene'-J-(Alkylene-O) n -Alkylene-, -(alkylene-O) n -Alkylene-J-alkylene'-, -J-(alkylene-O) n -Alkylene-, -(alkylene-O) n -Alkylene-J-(Alkylene-O) n '-Alkylene-J'-, -W-, -Alkylene-W-, Alkylene'-J- (Alkylene-NMe) n -Alkylene-W-, -J-(Alkylene-NMe) n -Alkylene-W-, -(Alkylene-O) n -Alkylene-UC(O)-, -(Alkylene-O) n -alkylene-U-alkylene; -J-alkylene-NMe-alkylene'-NMe-alkylene''-W-, and -alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene'''-W-; W has the following structure:

[0090] [ka]

[0091] U has the structure:

[0092] [ka]

[0093] Each J and J' independently has the structure:

[0094] [ka]

[0095] n and n' are each independently an integer of 1 or greater; D has the following structure:

[0096] [ka]

[0097] Each R 17 are independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkyleneoxide, substituted polyalkyleneoxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R")2, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-SS-(aryl or substituted aryl), -C(O)R", -C(O)R", or -C(O)N(R")2, wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl; Each Z1 is a bond, CR 17 R 17,O,S,NR',CR 17 R 17 -CR 17 R 17 , C.R. 17 R 17 -O, O-CR 17 R 17 , C.R. 17 R 17 -S, S-CR 17 R 17 , C.R. 17 R 17 -NR' or NR'-CR 17 R 17 and each R' is H, alkyl, or substituted alkyl; each Z2 is selected from the group consisting of a bond, -C(O)-, -C(S)-, optionally substituted alkylene having 1 to 3 carbon atoms, optionally substituted alkenylene having 1 to 3 carbon atoms, and optionally substituted heteroalkyl; each Z3 is selected from the group consisting of a bond, optionally substituted alkylene having 1 to 4 carbon atoms, optionally substituted alkenylene having 1 to 4 carbon atoms, optionally substituted heteroalkyl, -O-, -S-, -C(O)-, -C(S)-, and -N(R')-; each T3 is a bond, C(R")(R"), O, or S (provided that when T3 is O or S, R" is not a halogen); each R" is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; m and p are 0, 1, 2, or 3 (provided that at least one of m or p is not 0); M2 is

[0098] [ka]

[0099] (wherein (a) indicates a bond to the B group, and (b) indicates a bond to each position on the heterocyclic group), M3 is

[0100] [ka]

[0101] (wherein (a) indicates a bond to the B group, and (b) indicates a bond to each position on the heterocyclic group), The M4 is

[0102] [ka]

[0103] (wherein (a) indicates the bond to the B group, and (b) indicates the bond to the respective position on the heterocyclic group), Each R 19 are independently selected from the group consisting of alkyl having 1 to 6 carbon atoms, alkoxy having 1 to 6 carbon atoms, ester, ether, thioether, aminoalkyl, halogen, alkyl ester, aryl ester, amide, aryl amide, alkyl halide, alkylamine, alkylsulfonic acid, alkylnitro, thioester, sulfonyl ester, halosulfonyl, nitrile, alkylnitrile, and nitro; q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11; Each R 16 are independently selected from the group consisting of hydrogen, halogen, alkyl, NO, CN, and substituted alkyl. In some embodiments, R1 is a polypeptide. In certain embodiments, the polypeptide is an antibody. In certain embodiments, the antibody is Herceptin. In other embodiments, R2 is a polypeptide. In certain embodiments, the polypeptide is an antibody. In certain embodiments, the antibody is Herceptin.

[0104] In some embodiments, compounds according to formula (XXXI-A) are described.

[0105] [ka]

[0106] In one embodiment, a pharmaceutical composition is described comprising any of the compounds described above and a pharmaceutically acceptable carrier, excipient, or binder.

[0107] In a further or alternative embodiment, a method for detecting the presence of a polypeptide in a patient comprises administering a polypeptide comprising at least one heterocycle-containing unnatural amino acid, wherein the heterocycle-containing unnatural amino acid polypeptide alters the immunogenicity of the polypeptide relative to a cognate natural amino acid.

[0108] It is understood that the methods and compositions described herein are not limited to, and may vary in, the methodology, protocols, cell lines, constructs, and reagents described herein, and that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the methods and compositions described herein, which are limited only by the appended claims.

[0109] As used in this specification and the appended claims, terms in the singular, whether first or second occurrences, include the plural references unless the context clearly dictates that only the singular reference is intended.

[0110] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Regarding the methods, devices, and materials used in practicing or testing the invention described herein, any methods, devices, and materials similar or equivalent to those described herein can be used, but the following describes the preferred methods, devices, and materials.

[0111] The publications and patent documents cited herein describe, for example, concepts and methodologies that may be used in connection with the present invention. For purposes of describing these concepts and methodologies, the entire contents of these publications and patent documents are incorporated herein by reference. The publications discussed herein are presented solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the inventors described herein are not entitled, by virtue of prior art or for any other reason, to status as inventors prior to such disclosure.

[0112] The term "aldol-based coupling" or "mixed aldol-based coupling" refers to the acid- or base-catalyzed condensation of a carbonyl compound with the enolate / enol of another carbonyl compound, which may be the same or different, to produce a β-hydroxy carbonyl compound (i.e., an aldol).

[0113] As used herein, the term "affinity label" refers to a label that reversibly or irreversibly binds to another molecule to modify, destroy, or form a compound with it. By way of example, affinity labels include enzymes and their substrates, or antibodies and their antigens.

[0114] The terms "alkoxy," "alkylamino," and "alkylthio" (or thioalkoxy) are used in the conventional sense to refer to alkyl groups linked to the molecule through an oxygen atom, an amino group, or a sulfur atom, respectively.

[0115] Unless otherwise specified, the term "alkyl," alone or as part of another molecule, may be fully saturated, monounsaturated, or polyunsaturated, and may refer to any alkyl group having the specified number of carbon atoms (i.e., C1-C6). 10"(C)" refers to a linear, branched, or cyclic hydrocarbon group, or combinations thereof, which can include divalent and polyvalent groups having 1 to 10 carbons (where "C" means 1 to 10 carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, and cyclopropylmethyl, as well as homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, and n-octyl. Unsaturated alkyl groups are those containing one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-propynyl, 3-propynyl, 3-butynyl, higher homologs, and higher isomers. The term "alkyl," unless otherwise noted, is also meant to include those derivatives of alkyl described in more detail herein, such as "heteroalkyl," "haloalkyl," and "homoalkyl."

[0116] The term "alkylene" alone or as part of another molecule refers to the radical (-CH2-) n (where n can be a number from 1 to about 24). By way of example, such groups include, but are not limited to, groups containing 10 or fewer carbon atoms, such as structures like -CHCH- and -CHCHCHCH-. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, typically containing 8 or fewer carbon atoms. The term "alkylene" is also intended to include groups described herein as "heteroalkylene," unless otherwise specified.

[0117] The term "amino acid" refers to natural and unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to natural amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), pyrrolysine, and selenocysteine. Amino acid analogs refer to compounds whose basic chemical structure is identical to that of natural amino acids. Examples include an alpha carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group. Such amino acid analogs may have the same basic chemical structure as natural amino acids, but with modified R groups (e.g., norleucine) or modified peptide backbones. Non-limiting examples of amino acid analogs include homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium.

[0118] Amino acids may be referred to herein by their names or by the three-letter or one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission, and nucleotides may be referred to by their commonly accepted single-letter codes.

[0119] An "amino terminal modification group" refers to any molecule capable of being attached to a terminal amine group. By way of example, such a terminal amine group may be located at the end of a polymer (including, but not limited to, polypeptides, polynucleotides, and polysaccharides). Terminal modification groups include, but are not limited to, various water-soluble polymers, peptides, or proteins. By way of example, terminal modification groups include polyethylene glycol or serum albumin. Terminal modification groups may also be used to modify the therapeutic properties of the polymer. Such modifications include, but are not limited to, increasing the blood half-life of the peptide.

[0120] "Antibody fragment" refers to any form of an antibody other than the full-length form. Antibody fragments herein include antibodies that are smaller components present in a full-length antibody and antibodies that have been modified. Antibody fragments include, but are not limited to, Fv, Fc, Fab, (Fab')2, single-chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, CDR combinations, variable regions, framework regions, constant regions, heavy chains, light chains, variable regions, alternative scaffolding non-antibody molecules, and bispecific antibodies (Maynard & Georgiou, 2000, Annu. Rev. Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol. 9:395-402). Another functional substructure is the single-chain Fv (scFv), which consists of the variable regions of immunoglobulin heavy and light chains covalently linked by a peptide linker (Sz Hu et al., 1996, Cancer Research, 56, 3055-3061). Typically, these small (Mr 25,000) proteins maintain specificity and affinity for the antigen in a single polypeptide and can therefore provide useful building blocks for constructing larger, antigen-specific molecules. Unless otherwise specified, any description or claim using the term "antigen" (singular or plural) is intended to specifically include "antigen fragments" (singular and plural).

[0121] As used herein, "antibody-drug conjugate" or "antibody-drug conjugate (ADC)" refers to an antibody molecule or fragment thereof that is covalently attached to one or more biologically active molecules. The biologically active molecules may be attached to the antibody via a linker, polymer, or other covalent bond.

[0122] As used herein, the terms "aromatic" or "aryl" refer to a closed ring structure having at least one ring with a bonded π-electron system, and include both carbocyclic and heterocyclic aryl groups (or "heteroaryl groups" or "heteroaromatic groups"). A carbocyclic or heterocyclic aromatic group can contain from about 5 to about 20 ring atoms. The terms include covalently bonded monocyclic or polycyclic fused ring groups (i.e., rings sharing adjacent pairs of carbon atoms). An aromatic group can be unsubstituted or substituted. Non-limiting examples of "aromatic" or "aryl" groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, anthracenyl, and phenanthracenyl. Substituents for each of the above-mentioned aryl and heteroaryl ring systems are selected from the group of acceptable substituents described herein.

[0123] Briefly, the term "aryl," when used in combination with other terms (including, but not limited to, aryloxy, arylthioxy, and aralkyl), includes both aryl and heteroaryl rings as defined above. Thus, the terms "aralkyl" or "alkaryl" are intended to include radicals in which an aryl group is attached to an alkyl group (including, but not limited to, benzyl, phenethyl, and pyridylmethyl), including alkyl groups in which a carbon atom (including, but not limited to, a methylene group) has been replaced with a heteroatom (such as, for example, an oxygen atom). Examples of such aryl groups include, but are not limited to, phenoxymethyl, 2-pyridyloxymethyl, and 3-(1-naphthyloxy)propyl.

[0124] As used herein, the term "arylene" refers to a divalent aryl radical. Non-limiting examples of "arylene" include phenylene, pyridinylene, pyrimidinylene, and thiophenylene. Substituents for arylene groups are selected from the group of acceptable substituents described herein.

[0125] A "bifunctional polymer," also referred to as a "bifunctional linker," refers to a polymer containing two functional groups capable of specifically reacting with another moiety to form a covalent or non-covalent bond. Such moieties include, but are not limited to, the side chain groups of natural or unnatural amino acids or peptides containing natural or unnatural amino acids. The other moieties that can be attached to a bifunctional linker or bifunctional polymer can be the same or different moieties. For example, a bifunctional linker has one functional group reactive with a group on a first peptide and another functional group reactive with a group on a second peptide, thereby forming a conjugate comprising the first peptide, the bifunctional linker, and the second peptide. Numerous techniques and linking molecules are known for attaching various compounds to peptides. See, for example, European Patent Application No. 188,256, U.S. Pat. No. 4,671,958, U.S. Pat. No. 4,659,839, U.S. Pat. No. 4,414,148, U.S. Pat. No. 4,699,784, U.S. Pat. No. 4,680,338, and U.S. Pat. No. 4,569,789. The entire contents of the above patent applications and patents are incorporated herein by reference. A "multifunctional polymer," also referred to as a "multifunctional linker," refers to a polymer containing two or more functional groups capable of reacting with other moieties. Such moieties include, but are not limited to, side groups of natural or unnatural amino acids or peptides containing natural or unnatural amino acids, which form covalent or noncovalent bonds. Bifunctional or multifunctional polymers (including, but not limited to, amino acid side groups) may be of any desired length or molecular weight and may be selected to provide a particular desired spacing or conformation between one or more molecules to be bound to a compound and the molecule or compound to which they are bound.

[0126] As used herein, the term "bioavailability" refers to the rate and extent to which a substance or its active moiety is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the systemic circulation. Increased bioavailability indicates an increase in the rate and extent to which a substance or its active moiety is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the systemic circulation. As an example, increased bioavailability may be indicated by increased blood levels of a substance or its active moiety relative to other substances or other active moieties. Non-limiting examples of methods for assessing increased bioavailability are provided in Examples 21-25. Such methods may be used to assess the bioavailability of any polypeptide.

[0127] As used herein, the terms "biologically active molecule," "biologically active moiety," or "bioactive agent" refer to any substance capable of affecting the physical or biochemical properties of a biological system, pathway, molecule, or interaction in an organism (including, but not limited to, viruses, bacteria, bacteriophages, transposons, prions, insects, fungi, plants, animals, and humans). In particular, as used herein, a bioactive molecule includes, but is not limited to, any substance intended for the diagnosis, cure, mitigation, treatment, or prevention of disease in humans or other animals, or that improves the physical or mental well-being of humans or animals. Examples of bioactive molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, hard drugs, soft drugs, prodrugs, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells, viruses, liposomes, microparticles, and micelles. Classes of biologically active molecules suitable for use in conjunction with the methods and compositions described herein include, but are not limited to, drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, bactericides, antivirals, anti-inflammatory agents, antitumor agents, cardiovascular agents, anxiolytics, hormones, growth factors, steroids, and bacterial toxins.

[0128] "Modulating biological activity" means increasing or decreasing the reactivity of a polypeptide, altering the selectivity of a polypeptide, or improving or decreasing the substrate selectivity of a polypeptide. Analysis of modified biological activity can be performed by comparing the biological activity of the non-naturally occurring polypeptide with the biological activity of the naturally occurring polypeptide.

[0129] As used herein, the term "biomaterial" refers to biologically derived materials, including, but not limited to, materials obtained from bioreactors and / or recombinant methods and techniques.

[0130] As used herein, the term "biophysical probe" includes probes capable of detecting or observing structural changes in molecules. Such molecules include, but are not limited to, proteins. "Biophysical probes" may also be used to detect or observe interactions between proteins and other macromolecules. Examples of biophysical probes include, but are not limited to, spin labels, fluorescent probes, and photoactivatable groups.

[0131] As used herein, the term "biosynthetic" refers to any method that utilizes a translation system (either cellular or non-cellular) and that includes the use of at least one of the following elements: polynucleotides, codons, tRNAs, and ribosomes. By way of example, unnatural amino acids may be "biosynthetically incorporated" into unnatural amino acid polypeptides using methods and techniques described herein and in non-limiting Example 20 ("In vivo methods for producing polypeptides comprising unnatural amino acids"). Additionally, methods for selecting useful unnatural amino acids that can be "biosynthetically incorporated" into unnatural amino acid polypeptides are described in non-limiting Example 20.

[0132] As used herein, the term "biotin analog" (also referred to as "biotin mimetic") is any molecule other than biotin that binds with high affinity to avidin and / or streptavidin.

[0133] As used herein, the term "carbonyl" refers to a group containing a moiety selected from the group consisting of -C(O)-, -S(O)-, -S(O)2-, and -C(S)-, including, but not limited to, groups containing one ketone group, at least one aldehyde group, at least one ester group, at least one carboxylic acid group, and / or at least one thioester group. Such carbonyl groups encompass ketones, aldehydes, carboxylic acids, esters, and thioesters. Furthermore, such groups may be part of a linear, branched, or cyclic molecule.

[0134] The term "carboxy-terminal modification group" refers to any molecule that can be attached to a terminal carboxy group. By way of example, such a terminal carboxy group may be located at the end of a polymer (including, but not limited to, a polypeptide, a polynucleotide, and a polysaccharide). Terminal modification groups include, but are not limited to, various water-soluble polymers, peptides, or proteins. By way of example, terminal modification groups include polyethylene glycol or serum albumin. Terminal modification groups may also be used to modify the therapeutic properties of the polymer. Such modifications include, but are not limited to, extending the blood half-life of the peptide.

[0135] As used herein, the term "chemically cleavable group," also referred to as "chemically labile," refers to a group that is destroyed or cleaved upon exposure to an acid, a base, an oxidizing agent, a reducing agent, a chemical initiator, or a radical initiator.

[0136] As used herein, the term "chemiluminescent group" refers to a group that emits light as a result of a chemical reaction without the need for heat. As an example, luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) reacts with an oxidizing agent such as hydrogen peroxide (HO) in the presence of a substrate and a metal catalyst to produce an excited-state reaction product (3-aminophthalate (3-APA)).

[0137] As used herein, the term "chromophore" refers to a molecule that absorbs light in the visible, ultraviolet, or infrared wavelengths.

[0138] As used herein, the term "cofactor" refers to an atom or molecule essential for the action of a macromolecule. Cofactors include, but are not limited to, inorganic ions, coenzymes, proteins, or other factors necessary for enzyme activity. Examples include the heme in hemoglobin, the magnesium in chlorophyll, and metal ions found in proteins.

[0139] As used herein, "cofolding" refers to a refolding process, reaction, or method that uses at least two molecules that interact with each other to convert an unfolded or improperly folded molecule into a properly folded molecule. As an example, "cofolding" uses at least two polypeptides that interact with each other to convert an unfolded or improperly folded polypeptide into a native, properly folded polypeptide. Such polypeptides may contain natural amino acids and / or at least one unnatural amino acid.

[0140] As used herein, a "comparison window" refers to any segment of contiguous positions used to compare a sequence to a reference sequence of the same number of contiguous positions after the two sequences have been optimally aligned. Such contiguous positions include, but are not limited to, a group consisting of about 20 to about 600 contiguous units (including about 50 to about 200 sequence units, and including about 100 to about 150 sequence units). By way of example, such sequences include polypeptides and polypeptides containing unnatural amino acids, and the contiguous units include, but are not limited to, natural and unnatural amino acids. Further, by way of example, such sequences include polynucleotides consisting of nucleotides corresponding to the contiguous units. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be achieved using local homology algorithms (Smith and Waterman, 2004). (1970) Adv. Appl. Math. 2:482c), homologous registration algorithms (Needleman and Wunsch (1970) J. Mol. Biol. 48:443), similarity search methods (Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444), computer implementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0141] By way of example, algorithms that can be used to determine percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al. (1997) Nuc. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. The BLAST algorithm variables W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as default a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915) alignment (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLAST algorithm is typically run with the "low complexity" filter turned off.

[0142] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which measures the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability when comparing a test nucleic acid to a reference nucleic acid is less than about 0.2, less than about 0.01, or less than about 0.001.

[0143] The term "conservatively modified variants" applies to both natural and non-natural amino acids, natural and non-natural nucleic acid sequences, and combinations thereof. With respect to a particular nucleic acid sequence, "conservatively modified variants" refers to natural and non-natural nucleic acids that encode identical or essentially identical natural and non-natural amino acid sequences. Alternatively, where natural and non-natural nucleic acids do not encode natural and non-natural amino acid sequences, "conservatively modified variants" refer to essentially identical sequences. As an example, due to the degeneracy of the genetic code, any particular protein can be encoded by a large number of nucleic acids with the same function. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variants are "silent variants," which are one type of conservatively modified variant. Thus, as an example, all natural or non-natural nucleic acid sequences herein that encode natural or non-natural polypeptides also describe all possible silent variations in natural or non-natural nucleic acids. Those skilled in the art will recognize that each codon (except AUG and TGG, which are normally the only codons that encode methionine and TGG are normally the only codons that encode tryptophan) of natural or non-natural nucleic acids can be modified to produce a functionally identical molecule. Accordingly, each silent variation of natural and non-natural nucleic acids that encode natural and non-natural polypeptides is implicit in each described sequence.

[0144] With respect to amino acid sequences, individual substitutions, deletions, or additions to nucleic acid, peptide, polypeptide, or protein sequences that result in the alteration, addition, or deletion of a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants" when such alterations result in the deletion, addition, or substitution of an amino acid with a chemically similar amino acid, either natural or unnatural. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the methods and compositions described herein.

[0145] Conservative substitution tables providing functionally similar amino acids are well known to those skilled in the art. The following eight groups contain amino acids that are conservative substitutions for one another: (1) alanine (A), glycine (G); (2) aspartic acid (D), glutamic acid (E); (3) asparagine (N), glutamine (Q); (4) arginine (R), lysine (K); (5) isoleucine (I), leucine (L), methionine (M), valine (V); (6) phenylalanine (F), tyrosine (Y), tryptophan (W); (7) serine (S), threonine (T); and (8) Cysteine ​​(C), Methionine (M) (See, e.g., Creighton, Proteins: Structure and Molecular Properties (W.H. Freeman & Co.; 2nd edition (December 1993).

[0146] The terms "cycloalkyl" and "heterocycloalkyl," when used alone or in combination with other terms, refer to cyclic versions of "alkyl" and "heteroalkyl," respectively, unless otherwise specified. Thus, cycloalkyl or heterocycloalkyl includes saturated, partially saturated, and fully saturated ring bonds. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Heteroatoms may include, but are not limited to, oxygen, nitrogen, or sulfur. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, and 2-piperazinyl. Furthermore, the term encompasses polycyclic structures, including, but not limited to, bicyclic and tricyclic ring structures. Similarly, the term "heterocycloalkylene," alone or as part of another molecule, refers to a divalent radical derived from a heterocycloalkyl. The term "cycloalkylene," alone or as part of another molecule, refers to a divalent radical derived from a cycloalkyl.

[0147] As used herein, the term "cyclodextrin" refers to a cyclic carbohydrate composed of at least six to eight glucose molecules arranged in a ring. The outer portion of the ring contains water-soluble groups. The central portion of the ring is a relatively nonpolar cavity capable of accommodating small molecules.

[0148] As used herein, the term "cytotoxic" refers to a compound that inflicts damage on cells.

[0149] As used herein, "denaturant" refers to any compound or material that causes reversible unfolding of a polymer. As an example, a "denaturant" may cause reversible unfolding of a protein. The merits of a denaturant will be determined by both the characteristics and concentration of the particular denaturant. By way of example, denaturants include, but are not limited to, chaotropes, detergents, organics, water-miscible solvents, phospholipids, or combinations thereof. Non-limiting examples of chaotropes include, but are not limited to, urea, guanidine, and sodium thiocyanate. Non-limiting examples of detergents include, but are not limited to, strong detergents such as sodium dodecyl sulfate or polyoxyethylene ethers (e.g., Tween or Triton detergents), Sarkosyl, weak non-ionic detergents (e.g., digitonin), weak cationic detergents such as N-2,3-(dioleoxy)-propyl-N,N,N-trimethylammonium, weak ionic detergents (e.g., sodium cholate or sodium deoxycholate), or zwitterionic detergents (including, but not limited to, sulfobetaine (an amphoteric detergent), 3-(3-chloroamidopropyl)dimethylammonio-1-propanesulfate (CHAPS), and 3-(3-chloroamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfate (CHAPSO)). Non-limiting examples of organic water-miscible solvents include, but are not limited to, acetonitrile, lower alkanols (particularly C2-C4 alkanols such as ethanol or isopropanol), or lower alkanediols (C2-C4 alkanediols such as ethylene glycol). The above non-limiting examples of organic water-miscible solvents may also be used as denaturing agents. Non-limiting examples of phospholipids include, but are not limited to, natural phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol, or synthetic phospholipid derivatives or variants such as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

[0150] As used herein, the term "desired functional group" refers to any group selected from the following: a label; a dye; a polymer; a water-soluble polymer; a polyethylene glycol derivative; a photocrosslinker; a cytotoxic compound; a drug; an affinity label; a photoaffinity label; a photoaffinity label; a reactive compound; a resin; a second protein analog, polypeptide analog, or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; DNA; RNA; an antisense polynucleotide; a polysaccharide; a water-soluble dendrimer; a cyclodextrin; a biomaterial; a nanoparticle; a spin label; a fluorescent probe; a metal-containing moiety; a radioactive moiety; a novel functional group; a group that interacts covalently or noncovalently with other molecules; a photocaged moiety; an actinic radiation-excitable moiety; a ligand; a photoisomerizable moiety; biotin; a biotin analog; a moiety incorporating a heavy element; or a chemically cleavable moiety. and any combination thereof. Examples of suitable non-natural amino acid polypeptides include: photocleavable groups; extended side chains; carbon-linked sugars; redox-active agents; aminothioacids; toxic moieties; isotopically labeled moieties; biophysical probes; phosphorescent groups; chemiluminescent groups; electron-dense groups; magnetic groups; intercalating groups; chromophores; energy transfer agents; biologically active agents (wherein the biologically active agent can include an agent having therapeutic activity, and the non-natural amino acid polypeptide or modified non-natural amino acid can act as a means to deliver a co-therapeutic or therapeutic agent to which the therapeutic agent is attached to a desired location in an organism); detectable labels; small molecules; inhibitory ribonucleic acids; radionucleotides; neutron capture agents; biotin derivatives; quantum dots; nanotransmitters; radioactive transmitters; enzyme antibodies; activated conjugate active agents; viruses; adjuvants; aggrecan; allergens; angiostatin; antihormones; antioxidants; aptamers; guide RNAs; saponins; shuttling vectors; macromolecules; mimotopes; receptors; reverse micelles; and any combination thereof.

[0151] As used herein, the term "diamine" refers to a group / molecule containing at least two amine functional groups, including, but not limited to, hydrazine, amidine, imine, 1,1-diamine, 1,2-diamine, 1,3-diamine, and 1,4-diamine groups. Furthermore, such groups may be part of a linear, branched, or cyclic molecule.

[0152] As used herein, the term "detectable label" refers to a label that can be observed using analytical techniques (including, but not limited to, fluorescence, chemiluminescence, electron spin resonance, ultraviolet-visible absorbance spectroscopy, mass spectrometry, nuclear magnetic resonance, magnetic resonance, and electrochemical methods).

[0153] The term "dicarbonyl" refers to a group containing at least two moieties selected from the group consisting of -C(O)-, -S(O)-, -S(O)2-, and -C(S)-. Such groups include, but are not limited to, 1,2-dicarbonyl, 1,3-dicarbonyl, 1,4-dicarbonyl groups, and groups containing at least one ketone group, at least one aldehyde group, at least one ester group, at least one carboxylic acid group, and / or at least one thioester group. Such dicarbonyl groups include diketone, ketoaldehyde, ketoacid, ketoester, and ketothioester groups. Furthermore, such groups may be part of linear, branched, or cyclic molecules. The two moieties of the dicarbonyl group may be the same or different, and either may contain a substituent that forms an ester, ketone, aldehyde, thioester, or amide, for example.

[0154] As used herein, the term "drug" refers to any substance used in the prevention, diagnosis, mitigation, treatment, or cure of a disease or condition.

[0155] As used herein, the term "dye" refers to a soluble color-forming agent that contains a chromophore.

[0156] As used herein, the term "effective amount" refers to that amount of an administered drug or compound sufficient to alleviate to some extent one or more symptoms of the disease or condition being treated. This may result in a reduction and / or alleviation of the signs, symptoms, or causes of a disease, or other desired alteration of a biological system. By way of example, the administered drug or compound includes, but is not limited to, a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-amino acid polypeptide. Compositions containing such natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-natural amino acid polypeptides can be administered for prophylactic, augmentative, and / or therapeutic treatments. An appropriate "effective" amount in any individual case may be determined using techniques, such as a dose escalation study.

[0157] As used herein, the term "electron-dense group" refers to a group that causes scattering of electrons when irradiated with an electron beam. Such groups include, but are not limited to, ammonium molybdate, bismuth cadmium iodide subnitrate, 99%, carbohydrazide, ferric chloride hexahydrate, hexamethylenetetramine, 98.5%, indium trichloride anhydrous, lanthanum nitrate, lead acetate trihydrate, lead citrate trihydrate, lead nitrate, periodic acid, phosphomolybdic acid, phosphotungstic acid, potassium ferricyanide, potassium ferrocyanide, ruthenium red, silver nitrate, silver proteinate (Ag assay: 8.0-8.5%) "Strong", silver tetraphenylporphyrin (S-TPPS), sodium chloroaurate, sodium tungstate, thallium nitrate, thiosemicarbazide (TSC), uranyl acetate, uranyl nitrate, and vanadyl sulfate.

[0158] As used herein, the term "energy transfer agent" refers to a molecule that donates energy to or receives energy from other molecules. As an example, fluorescence resonance energy transfer (FRET) is a dipole-dipole coupling process in which the excited state energy of a fluorescent donor molecule is non-radiatively transferred to a non-excited acceptor molecule, which then fluoresces and emits the donated energy at a longer wavelength.

[0159] The term "enhance" or "enhancing" means to increase or prolong the effectiveness or duration of a desired effect. By way of example, "enhancing" the effect of a therapeutic agent refers to the ability to increase or prolong the effectiveness or duration of the effect of the therapeutic agent acting during the treatment of a disease, disorder, or condition. As used herein, an "enhancing-effective amount" refers to an amount adequate to enhance the effectiveness of the therapeutic agent in treating a disease, disorder, or condition. When used in a patient, the amount effective for such use will depend on the severity and course of the disease, disorder, or condition, previous treatments, the patient's health status and response to the drug, and the judgment of the treating physician.

[0160] As used herein, the term "eukaryote" refers to organisms that belong to the phylogenetic domain Eukarya, including, but not limited to, animals (including, but not limited to, mammals, insects, reptiles, birds, etc.), ciliates, plants (including, but not limited to, monocotyledons, dicotyledons, and algae), fungi, yeast, flagellates, microsporidia, and protists.

[0161] As used herein, the term "fatty acid" refers to a carboxylic acid having a hydrocarbon side chain of C6 or longer.

[0162] As used herein, the term "fluorophore" refers to a fluorescent molecule that emits a photon when excited.

[0163] As used herein, the terms "functional group," "active moiety," "activating group," "leaving group," "reactive site," "chemically reactive group," and "chemically reactive moiety" refer to a portion or unit of a molecule where a chemical reaction occurs. These terms are somewhat synonymous in the chemical arts and are used herein to refer to the portion of a molecule that provides a function or activity and reacts with other molecules.

[0164] The term "halogen" encompasses fluorine, chlorine, iodine, and bromine.

[0165] As used herein, the term "haloacyl" refers to an acyl group that contains a halogen moiety (including, but not limited to, -C(O)CH3, -C(O)CF3, and -C(O)CH2OCH3).

[0166] As used herein, the term "haloalkyl" refers to an alkyl group that contains a halogen moiety (including, but not limited to, -CF3 and -CH2CF3).

[0167] As used herein, the term "heteroalkyl" refers to a linear, branched, or cyclic hydrocarbon radical, or combinations thereof, composed of an alkyl group and at least one heteroatom selected from the group consisting of O, N, Si, and S, wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom(s) O, N, S, and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, and -CH=CH-N(CH3)-CH3. Additionally, up to two heteroatoms may be consecutive (such as, by way of example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3).

[0168] The term "heterocycle-based linkage" or "heterocyclic bond" refers to the moiety formed by reacting a dicarbonyl group with a diamine group. The resulting reaction product is a heterocycle containing a heteroaryl or heterocycloalkyl group. The resulting heterocyclic group serves as a chemical linkage between the non-natural amino acid or non-natural amino acid polypeptide and another functional group. In one embodiment, the heterocyclic bond comprises a nitrogen-containing heterocyclic bond, including, by way of example, a pyrazole bond, a pyrrole bond, an indole bond, a benzodiazepine bond, and a pyrazolone bond.

[0169] Similarly, the term "heteroalkylene" refers to a divalent radical derived from a heteroalkyl (exemplified, but not limited to, -CH-CH-S-CH-CH- and -CH-S-CH-CH-NH-CH-). For heteroalkylene groups, the same or different heteroatoms (including, but not limited to, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and aminooxyalkylene, etc.) can occupy either or both chain termini. Furthermore, for alkylene and heteroalkylene linking groups, no directionality of the linking group is implied by the written direction of the formula depicting the linking group. As an example, the formula -C(O)R'- represents both -C(O)R'- and -R'C(O)-.

[0170] As used herein, the term "heteroaryl" or "heteroaromatic" refers to an aryl group containing at least one heteroatom selected from N, O, and S. The nitrogen and sulfur atoms in the aryl group may optionally be oxidized, and the nitrogen atoms may optionally be quaternized. A heteroaryl group may be substituted or unsubstituted. A heteroaryl group may be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of such heteroaryl groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.

[0171] As used herein, the term "homoalkyl" refers to an alkyl group that is a hydrocarbon group.

[0172] As used herein, the term "identical" refers to two or more identical sequences or subsequences. Furthermore, as used herein, the term "substantially identical" refers to two or more sequences that, when compared and aligned for maximum correspondence over a comparison window or designated region, have a certain percentage of identical contiguous units, as measured by a comparison algorithm or by manual alignment and visual inspection. As an example, two or more sequences may be "substantially identical" if the identity of contiguous units over a specified region is about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. Such percentages of identity describe the "percent identity" of two or more sequences. Sequence identity can exist over a region of at least about 75-100 contiguous units in length, over a region of about 50 contiguous units in length, or, if not specified, over the entire sequence. This definition also refers to the complement of a test sequence. For example, two or more polypeptide sequences are identical if their amino acid residues are identical. On the other hand, two or more polypeptide sequences are "substantially identical" if the identity of amino acid residues across a specific region is about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. This identity can exist across a region of at least about 75 to about 100 amino acids in length, across a region of about 50 amino acids in length, or across the entire polypeptide sequence in any particular case. Furthermore, for example, two or more polynucleotide sequences are identical if their nucleic acid residues are identical. On the other hand, two or more polynucleotide sequences are "substantially identical" if the identity of nucleic acid residues across a specific region is about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. The identity can exist over a region of at least about 75 to about 100 nucleic acids in length, over a region of about 50 nucleic acids in length, or in certain cases over the entire sequence of the nucleotide sequence.

[0173] In sequence comparison, one sequence usually serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program variables are designated. Default sequence algorithm program variables can be used. Alternatively, alternative variables can be designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program variables.

[0174] As used herein, the term "immunogenicity" refers to the antibody response to administration of a therapeutic agent. Quantitative and qualitative assays for detecting anti-non-natural amino acid polypeptide antibodies in biological fluids can be used to determine immunogenicity to a therapeutic non-natural amino acid polypeptide. These assays include, but are not limited to, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), luminescence immunoassays (LIA), and fluorescence immunoassays (FIA). Detecting immunogenicity to a therapeutic non-natural amino acid polypeptide involves comparing the antibody response after administration of the therapeutic non-natural amino acid polypeptide with the antibody response after administration of the therapeutic natural amino acid polypeptide.

[0175] As used herein, the term "intercalating agent," also referred to as an "intercalating group," refers to a chemical substance capable of inserting into intramolecular or intermolecular spaces. As an example, the intercalating agent or intercalating group may be a DNA molecule. It may be a molecule that inserts between stacked bases of double-stranded DNA.

[0176] As used herein, the term "isolated" refers to the separation and removal of a target element from non-target elements. An isolated material can be dry or semi-dry, or in solution (including, but not limited to, aqueous solutions). An isolated element can be homogeneous or part of a pharmaceutical composition with additional pharmaceutically acceptable carriers and / or excipients. Purity and homogeneity may be determined using analytical chemistry techniques (including, but not limited to, polyacrylamide gel electrophoresis or high performance liquid chromatography). Furthermore, when a target element is isolated and is the predominant species present in a preparation, the element is described herein as substantially purified. As used herein, the term "purified" can refer to a target element that is at least 85% pure, at least 90% pure, at least 95% pure, or at least 99% pure or greater. As an example, a nucleic acid or protein is "isolated" if it is free from at least some of the cellular elements with which it is naturally associated. Alternatively, the nucleic acid or protein is "isolated" if it is condensed to a level greater than that obtainable by in vitro or in vivo production. Further, as an example, a gene is isolated if it is separated from open reading frames that flank it and encode proteins other than the gene of interest.

[0177] As used herein, the term "label" refers to a substance that is incorporated into a compound and is easily detected. The physical distribution of said substance may be detected and / or monitored.

[0178] As used herein, the term "linkage" refers to a bond or chemical moiety that results from a chemical reaction between a functional group of a linker and another molecule. Such bonds may include, but are not limited to, covalent and non-covalent bonds. Furthermore, such chemical moieties include, but are not limited to, esters, carbonates, imine phosphate esters, hydrazones, acetals, orthoesters, peptide bonds, and oligonucleotide linkages. A hydrolytically stable linkage refers to a bond that exhibits substantial stability in water and does not react with water at useful pH values ​​(e.g., under physiological conditions for extended periods or indefinitely). A hydrolytically unstable or degradable linkage means that the linkage is degradable in water or aqueous solutions (including, for example, blood). An enzyme-labile or degradable linkage means that the linkage is degradable by one or more enzymes. By way of example, PEG and related polymers may contain degradable linkages within the polymer backbone or within the linking group between the polymer backbone and one or more of the polymer's terminal functional groups. Such degradable linkages include, but are not limited to, ester linkages formed by reaction of a PEG carboxylic acid or an activated PEG carboxylic acid with an alcohol group on a bioactive agent. It should be noted that such ester groups generally hydrolyze under physiological conditions to release the bioactive agent. Other hydrolytically degradable bonds include, but are not limited to, carbonate bonds; imine bonds formed from the reaction of an amine and an aldehyde; phosphate ester bonds formed by reacting an alcohol with a phosphate group; hydrazone bonds, which are the reaction product of a hydrazide and an aldehyde; acetal bonds, which are the reaction product of an aldehyde and an alcohol; orthoester bonds, which are the reaction product of a formate and an alcohol; peptide bonds formed by an amine group (including, but not limited to, an amine group located at the end of a polymer such as PEG) and a carboxyl group of a peptide; and oligonucleotide bonds formed from a phosphoramidite group (including, but not limited to, a phosphoramidite group at the end of a polymer) and a 5' hydroxyl group of an oligonucleotide.

[0179] As used herein, the term "culture medium" or "media" refers to any culture medium used for the growth and recovery of cells and / or the expression and / or secretion of products by such cells. Such "culture medium" or "media" includes, but is not limited to, any host cell (e.g., bacterial host cell, yeast host cell, insect host cell, plant host cell, eukaryotic host cell, mammalian host cell, CHO cell, prokaryotic host cell, E. coli host cell, or Pseudomonas host cell) and a solution, solid, semi-solid, or solid support that may hold or contain cellular contents. Such "culture medium" or "media" includes, but is not limited to, the medium or broth in which host cells are cultured and polypeptides are secreted (either before or after the growth stage). Such "culture medium" or "media" also includes, by way of example only, buffers or reagents containing host cell lysates in which polypeptides are produced intracellularly and in which host cells are lysed or disrupted to release the polypeptides.

[0180] As used herein, the term "metabolite" refers, by way of example, to a derivative of a compound such as a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-natural amino acid polypeptide, by way of example, to a compound such as a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-natural amino acid polypeptide, produced when that compound is metabolized. The term "pharmaceutically active metabolite" or "active metabolite" refers, by way of example, to a biologically active derivative of a compound such as a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-natural amino acid polypeptide, by way of example, to a compound such as a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-natural amino acid polypeptide, produced when that compound is metabolized.

[0181] As used herein, the term "metabolized" refers to all of the processes by which a particular substance is transformed by an organism. Such processes include, but are not limited to, hydrolysis reactions and enzyme-catalyzed reactions. For further details regarding metabolism, see The Pharmacological Basis of Therapeutics, 9th Edition, McGraw-Hill (1996). By way of example, metabolic products of natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-natural amino acid polypeptides can be identified by administering the natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-natural amino acid polypeptides to a host and analyzing tissue samples from the host, or by incubating the natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-natural amino acid polypeptides with hepatocytes in vitro and analyzing the resulting compounds.

[0182] As used herein, the term "metal chelator" refers to a molecule that forms a metal complex with a metal ion. As an example, such a molecule may form two or more coordinate bonds with a central metal ion to form a ring structure.

[0183] As used herein, the term "metal-containing moiety" refers to a group that contains a metal ion, atom, or particle. Such moieties include, but are not limited to, cisplatin, chelated metal ions (such as nickel, iron, platinum, etc.), and metal nanoparticles (such as nickel, iron, platinum, etc.).

[0184] As used herein, the term "heavy atom-incorporating moiety" refers to a group that incorporates an ion of an atom usually heavier than carbon. Such ions or atoms include, but are not limited to, silicon, tungsten, gold, lead, and uranium.

[0185] As used herein, the term "modified" refers to the presence of an alteration to a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide. Such an alteration or modification can be obtained by post-synthetic modification of a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide, or by co- or post-translational modification of a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide. "Modified or unmodified" form means that the natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide being mentioned is optionally modified, i.e., the natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide being mentioned can be modified or unmodified.

[0186] As used herein, the term "modulated serum half-life" refers to a positive or negative change in the circulating half-life of a modified biologically active molecule compared to its unmodified form. Exemplary modified biologically active molecules include, but are not limited to, natural amino acids, non-natural amino acids, natural amino acid polypeptides, or non-natural amino acid polypeptides. Exemplary serum half-life is measured by collecting blood samples at various time points after administration of the biologically active molecule or modified biologically active molecule and determining the concentration of the molecule in each sample. The correlation between time and serum concentration allows for calculation of serum half-life. As an example of a modified serum half-life, an enhanced serum half-life can allow for improved dosing regimens or avoid toxic effects. Such serum enhancements can be at least about 2-fold, at least about 3-fold, at least about 5-fold, or at least about 10-fold. An exemplary, but non-limiting, method for assessing enhanced serum half-life is provided in Example 33. This method can be used to assess the serum half-life of any polypeptide.

[0187] As used herein, the term "modulated therapeutic half-life" refers to a positive or negative change in the half-life of a therapeutically effective amount of a modified biologically active molecule compared to its unmodified form. By way of example, the modified biologically active molecule may include, but is not limited to, a natural amino acid, a non-natural amino acid, a natural amino acid polypeptide, or a non-natural amino acid polypeptide. By way of example, the therapeutic half-life is measured by measuring the pharmacokinetic and / or pharmacological properties of the molecule at various time points after administration. An enhanced therapeutic half-life may enable a particularly beneficial dosing regimen or a particularly beneficial total dose, or may avoid undesirable effects. By way of example, an enhanced therapeutic half-life may result from enhanced efficacy, enhanced or reduced binding of the modified molecule to its target, enhancement or attenuation of other components or mechanisms of action of the unmodified molecule, or increased or decreased molecular degradation by enzymes such as proteases. An exemplary, but non-limiting, method for assessing enhanced therapeutic half-life is provided in Example 33. This method can be used to assess the therapeutic half-life of any polypeptide.

[0188] As used herein, the term "nanoparticle" refers to particles having a particle size of about 500 nm to about 1 nm.

[0189] As used herein, the term "nearly stoichiometric" refers to a molar ratio of compounds being used in a chemical reaction of about 0.75 to about 1.5.

[0190] As used herein, the term "non-eukaryote" refers to a non-eukaryotic organism. By way of example, the non-eukaryotic organism can belong to the Eubacteria (including, but not limited to, Escherichia coli, Thermus thermophilus, or Bacillus stearothermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida, etc.) (phylogenetic domain), or the Archaea (including, but not limited to, Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, or Halobacterium (e.g., Haloferax volcanii and Halobacterium species NRC-1)) (or phylogenetic region).

[0191] A "non-natural amino acid" refers to an amino acid that is not one of the 20 common amino acids and is not pyrrolysine or selenocysteine. Other terms that may be used synonymously with the term "non-natural amino acid" include "non-naturally encoded amino acid," "unnatural amino acid," "non-naturally occurring amino acid," and various hyphenated and non-hyphenated forms thereof. The term "non-natural amino acid" includes, but is not limited to, amino acids that arise naturally by modification of a naturally encoded amino acid (including, but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine) and are not incorporated into a growing polypeptide chain by the translation complex. Examples of naturally occurring amino acids that are not naturally encoded include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine. Furthermore, the term "non-natural amino acid" includes, but is not limited to, amino acids that do not occur in nature and are obtained synthetically or by modification of non-natural amino acids.

[0192] As used herein, the term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides, and polymers thereof in single- or double-stranded form. By way of example, such nucleic acids and nucleic acid polymers include, but are not limited to, (i) analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides, (ii) analogs of oligonucleotides (PNA (peptide nucleic acid)), analogs of DNA used in antisense technology (such as phosphorothioates and phosphoramidates), and (iii) conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences and sequences explicitly indicated. As an example, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0193] As used herein, "oxidizing agent" refers to a compound or substance that can remove electrons from a compound to be oxidized. Exemplary oxidizing agents include, but are not limited to, oxidized glutathione, cystine, cystamine, oxidized dithiothreitol, oxidized erythreitol, and oxygen. A wide variety of oxidizing agents are suitable for use in the methods and compositions described herein.

[0194] As used herein, the term "pharmaceutically acceptable" refers to, but is not limited to, salts, carriers, diluents, and the like that do not interfere with the biological activity or properties of the compound and that are relatively non-toxic. That is, it refers to a substance that may be administered to an individual without causing undesired biological effects or interacting in a manner that degrades any of the components of the composition in which it is contained.

[0195] As used herein, the term "photoaffinity label" refers to a label with a group that, when illuminated with light, forms a bond with a molecule for which the label has an affinity. By way of example, such a bond may be covalent or non-covalent.

[0196] As used herein, the term "photocaged moiety" refers to a group that, upon illumination with a particular wavelength, covalently or non-covalently binds other ions or molecules.

[0197] As used herein, the term "photocleavable group" refers to a group that is cleaved when exposed to light.

[0198] As used herein, the term "photocrosslinker" refers to a compound containing two or more functional groups that react upon exposure to light to form covalent or non-covalent bonds with two or more monomeric molecules or macromolecules.

[0199] As used herein, the term "photoisomerizable moiety" refers to a group that changes from one isomeric form to another isomeric form when struck by light.

[0200] As used herein, the term "polyalkylene glycol" refers to a linear or branched polymeric polyether polyol. Such polyalkylene glycols include, but are not limited to, polyethylene glycol, polypropylene glycol, polybutylene glycol, and derivatives thereof. Other exemplary embodiments are listed in commercial supplier catalogs, such as Shearwater Corporation's "Polyethylene Glycol and Derivatives for Biomedical Applications" (2001). By way of example, such polymeric polyether polyols have an average molecular weight between about 0.1 kDa and about 100 kDa. By way of example, such polymeric polyether polyols include, but are not limited to, those between about 100 Da and about 100,000 Da or more. The molecular weight of the polymer is between about 100 Da and about 100,000 Da, for example, about 100,000 Da, about 95,000 Da, about 90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da, about 45,000 Da, about 40,000 Da, about 35,000 Da, about 30,000 Da, about 25,000 Da, Examples of molecular weights include, but are not limited to, about 20,000 Da, about 15,000 Da, about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, about 1,000 Da, about 900 Da, about 800 Da, about 700 Da, about 600 Da, about 500 Da, 400 Da, about 300 Da, about 200 Da, and about 100 Da. In some embodiments, the molecular weight of the polymer is between about 100 Da and about 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 100 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 2,000 Da and about 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 5,000 Da and about 40,000 Da.In some embodiments, the molecular weight of the polymer is between about 10,000 Da and about 40,000 Da. In some embodiments, the poly(ethylene glycol) molecule is a branched polymer. The molecular weight of the branched PEG is between about 1,000 Da and about 100,000 Da, about 100,000 Da, about 95,000 Da, about 90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da, about 45,000 Da, about 40,000 Da, and the like. Examples of branched PEG include, but are not limited to, about 35,000 Da, about 30,000 Da, about 25,000 Da, about 20,000 Da, about 15,000 Da, about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, and about 1,000 Da. In some embodiments, the molecular weight of the branched PEG is between about 1,000 Da and about 50,000 Da. In some embodiments, the molecular weight of the branched PEG is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the branched PEG is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the branched PEG is between about 5,000 Da and about 20,000 Da, hi other embodiments, the molecular weight of the branched PEG is between about 2,000 Da and about 50,000 Da.

[0201] As used herein, the term "polymer" refers to a molecule made up of repeating subunits, including, but not limited to, a polypeptide, a polynucleotide, or a polysaccharide, or a polyalkylene glycol.

[0202] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description directed to a polypeptide applies equally to a description of a peptide and a description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers and to amino acid polymers in which one or more amino acid residues are unnatural amino acids. Furthermore, such terms "polypeptide," "peptide," and "protein" encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are joined by covalent peptide bonds.

[0203] The term "post-translational modification" refers to any modification of a natural or unnatural amino acid that occurs to that amino acid after it is translationally incorporated into a polypeptide chain, including, but not limited to, in vivo co-translational modifications, in vitro co-translational modifications (e.g., in a cell-free translation system), in vivo post-translational modifications, and in vitro post-translational modifications.

[0204] As used herein, the term "prodrug" or "pharmaceutically acceptable prodrug" refers to a substance that is converted into the parent drug in vivo or in vitro, does not inhibit the biological activity or properties of the drug, and is relatively non-toxic; i.e., it refers to a substance that can be administered to an individual without causing undesirable biological effects or interacting in a way that degrades any of the components of the composition in which it is contained. Generally, a prodrug is a drug precursor that, following administration to a subject and subsequent absorption, is converted to an active or more active species through any process (e.g., conversion by a metabolic pathway). Some prodrugs have chemical groups present on the prodrug that reduce its activity and / or confer solubility or other properties on the drug. When the chemical group is cleaved and / or modified from the prodrug, the active drug is generated. Prodrugs are converted to active drugs by enzymatic or non-enzymatic reactions in the body. Prodrugs can provide improved physiochemical properties, such as improved solubility or improved transport properties that specifically target certain cells, tissues, organs, or ligands, and can also improve the therapeutic value of the drug. Advantages of such prodrugs include, but are not limited to, (i) easier administration than the parent drug, (ii) unlike the parent drug, prodrugs can be absorbed and utilized by the body by oral administration, and (iii) compared to the parent drug, prodrugs also have higher solubility in pharmaceutical compositions. Prodrugs include pharmacologically inactive or less active derivatives of active drugs. Prodrugs are intended to control the amount of a drug or biologically active molecule that reaches the intended site of action by manipulating the drug's physicochemical, biopharmaceutical, or pharmacokinetic properties. Examples of prodrugs include, but are not limited to, non-natural amino acid polypeptides that are administered as esters ("prodrugs") to facilitate transport across cell membranes where aqueous solubility adversely affects mobility, and then metabolically hydrolyzed to the carboxylic acid (active compound), where aqueous solubility is beneficial within the cell. Prodrugs are designed as reversible drug derivatives used as modulators to improve drug delivery to site-specific tissues.

[0205] As used herein, the term "prophylactically effective amount" refers to the amount of a composition comprising at least one non-natural amino acid polypeptide or at least one modified non-natural amino acid polypeptide administered prophylactically to a patient who will suffer to some extent one or more symptoms of the disease, condition, or disorder being treated. In such prophylactic uses, the amount will depend on the patient's state of health, weight, and the like. Determination of such prophylactically effective amounts by routine experimentation (including, but not limited to, a dose escalation clinical trial) is considered well within the skill of one in the art.

[0206] As used herein, the term "protected" refers to the presence of a "protecting group" or moiety that prevents reaction of a chemically reactive functional group under certain reaction conditions. The protecting group varies depending on the type of chemically reactive group being protected. As an example, (i) when the chemically reactive group is an amine or hydrazide, the protecting group is selected from the group consisting of tert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc); (ii) when the chemically reactive group is a thiol, the protecting group is orthopyridyl disulfide; and (iii) when the chemically reactive group is a carboxylic acid, such as butanoic acid or propionic acid, or a hydroxyl group, the protecting group can be a benzyl group or an alkyl group, such as methyl, ethyl, or tert-butyl.

[0207] In one example, the blocking / protecting group is

[0208] [ka]

[0209] may be selected from:

[0210] Further protecting groups include, but are not limited to, photolabile groups such as Nvoc and MeVoc, and other protecting groups known in the art, as described in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999, which is incorporated herein by reference in its entirety.

[0211] As used herein, the term "radioactive moiety" refers to a group having an atomic nucleus that spontaneously emits radiation such as alpha particles, beta particles, or gamma particles, where alpha particles are helium nuclei, beta particles are electrons, and gamma particles are high-energy photons.

[0212] As used herein, the term "reactive compound" refers to a compound that will react with another atom, molecule, or compound under the appropriate conditions.

[0213] The term "recombinant host cell," also referred to as "host cell," refers to a cell that contains an exogenous polynucleotide, regardless of the method used for insertion (e.g., direct uptake, transduction, f-mating, or other known methods used to create a recombinant host cell). By way of example, the exogenous polynucleotide may be a vector (e.g., a plasmid) that does not integrate into the host's genome, or may be integrated into the host's genome.

[0214] As used herein, the term "redox-active substance" refers to a molecule that oxidizes or reduces another molecule, thereby reducing or oxidizing the redox-active substance. Examples of redox-active substances include ferrocene, quinones, Ru, 2+ / 3+ Complex, Co 2+ / 3+ complex, and Os 2+ / 3+ These include, but are not limited to, complexes.

[0215] As used herein, the term "reducing agent" refers to a compound or substance that can add electrons to a compound in a reduced state. Exemplary reducing agents include, but are not limited to, dithiothreitol (DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine (2-aminoethanethiol), and reduced glutathione. Illustratively, such reducing agents maintain sulfhydryl groups in a reduced state and are used to reduce intramolecular or intermolecular disulfide bonds.

[0216] As used herein, "refolding" refers to any process, reaction, or method that transforms an improperly folded or unfolded state into a native or properly folded conformation. As an example, refolding transforms a polypeptide containing disulfide bonds from an improperly folded or unfolded state into a native or properly folded conformation with respect to the disulfide bonds. Such disulfide bond-containing polypeptides include natural amino acid polypeptides and non-natural amino acid polypeptides.

[0217] As used herein, the term "resin" refers to high molecular weight, insoluble polymer beads. By way of example, these beads are used as supports for solid-phase peptide synthesis or as a site to which molecules are attached prior to purification.

[0218] As used herein, the term "monosaccharide" refers to a series of carbohydrates, including, but not limited to, sugars, monosaccharides, oligosaccharides, and polysaccharides.

[0219] As used herein, the term "safety" or "safety" refers to the number of times the drug is administered relative to the potential side effects associated with the administration of the drug. As an example, a drug that produces few or no side effects even when administered frequently can be said to have excellent safety. An example of a method for evaluating safety is provided in Example 26, but is not limited thereto. This method can be used to evaluate the safety of any polypeptide.

[0220] As used herein, the phrases "selectively hybridize to" or "specifically hybridize to" refer to the binding, duplexing, or hybridization of a molecule with a particular nucleotide sequence under stringent hybridization conditions when the sequence is present in a complex mixture (including, but not limited to, whole cellular or library DNA or RNA).

[0221] As used herein, the term "spin label" refers to a molecule containing an atom or group of atoms (i.e., a stable paramagnetic group) that exhibits an unpaired electron spin that can be detected by electron spin resonance spectroscopy and attached to another molecule. Such spin-label molecules include, but are not limited to, nitrile radicals and nitroxides, and single or dual spin labels.

[0222] As used herein, the term "stoichiometric" refers to a molar ratio of compounds being used in a chemical reaction of about 0.9 to about 1.1.

[0223] As used herein, the term "stoichiometric-like" refers to a chemical reaction that becomes stoichiometric or near stoichiometric depending on changes in reaction conditions or the presence or absence of additives, including, but not limited to, increased temperature or pH. Such additives include, but are not limited to, promoters.

[0224] The term "stringent hybridization conditions" refers to the hybridization of sequences of DNA, RNA, PNA, or other nucleic acid mimetics, or combinations thereof, under conditions of low ionic strength and high temperature. For example, under stringent conditions, a probe will hybridize to the subsequence targeted by the probe in a complex mixture of nucleic acids (including, but not limited to, whole cells, a DNA library, or an RNA library), but will not hybridize to other sequences in the complex mixture. Stringent conditions are sequence-dependent, and different environments will result in different stringent conditions. For example, the longer the sequence, the higher the temperature required for specific hybridization. Stringent hybridization conditions include, but are not limited to, (i) about 5 to 10°C lower than the thermal melting temperature (Tm) of the specific sequence at a defined ionic strength and pH; (ii) about 0.01M to about 1.0M salt concentration at about pH 7.0 to about pH 8.3, and a temperature of at least about 30°C for short probes (including, but not limited to, probes of about 10 to about 50 nucleotides) and at least about 60°C for long probes (including, but not limited to, probes longer than 50 nucleotides); (iii) the addition of a destabilizing agent, such as, but not limited to, formamide; and (iv) incubation in 50% formamide, 5xSSC, and 1% SDS at 42°C or incubation in 5xSSC, 1% SDS at 65°C, followed by a wash in 0.2xSSC and about 0.1% SDS at 65°C for about 5 to about 120 minutes. By way of example, and not limitation, detection of selective or specific hybridization includes a positive signal that is at least two times background hybridization.Extensive guidance on nucleic acid hybridization is provided in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993).

[0225] As used herein, the term "subject" refers to an animal that has been the object of treatment, observation, or experiment. By way of example, and not limitation, a subject includes a mammal, such as a human.

[0226] As used herein, the term "substantially purified" refers to an element of interest that is substantially or substantially free from other elements that accompany or interact with the element of interest prior to purification. By way of example, an element of interest is "substantially purified" when a preparation of the element of interest contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating elements. Thus, a "substantially purified" element of interest has a purity of about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater. By way of example, a natural or non-natural amino acid polypeptide is purified from a naturally occurring cell or a host cell in the case of recombinantly producing the natural or non-natural amino acid polypeptide. By way of example, a preparation of natural or non-natural amino acid polypeptides is "substantially purified" when the preparation contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminants. By way of example, when the natural or non-natural amino acid polypeptide is recombinantly produced by a host cell, the natural or non-natural amino acid polypeptide is present at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less by dry weight of the cells. By way of example, when a natural or non-natural amino acid polypeptide is recombinantly produced by a host cell, the natural or non-natural amino acid polypeptide is present in the culture medium at about 5 g / L, about 4 g / L, about 3 g / L, about 2 g / L, about 1 g / L, about 750 mg / L, about 500 mg / L, about 250 mg / L, about 100 mg / L, about 50 mg / L, about 10 mg / L, or about 1 mg / L or less of the dry weight of the cells.By way of example, a "substantially purified" natural or non-natural amino acid polypeptide has a purity of about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more as determined by appropriate methods (e.g., but not limited to, SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis).

[0227] The term "substituents," also referred to as "non-interfering substituents," refers to groups that are used to substitute for other groups in a molecule. These groups include halo, C1-C 10 Alkyl, C2-C 10 Alkenyl, C2~ 10 Alkynyl, C1-C 10 Alkoxy, C5-C 12 Aralkyl, C3-C 12 Cycloalkyl, C4-C 12 Cycloalkenyl, phenyl, substituted phenyl, toluolyl, xylenyl, biphenyl, C2-C 12 Alkoxyalkyl, C5-C 12 Alkoxyaryl, C5-C 12 Aryloxyalkyl, C7-C 12 Oxyaryl, C1-C6 alkylsulfinyl, C1-C 10 Alkylsulfonyl, -(CH2) m -O-(C1~C 10 alkyl) (where m is 1 to 8), aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclic radical, substituted heterocyclic radical, nitroalkyl, -NO2, -CN, -NRC(O)-(C1-C 10 alkyl), -C(O)-(C1-C 10 alkyl), C2-C 10 Alkylthioalkyl, -C(O)O-(C1-C 10 alkyl), -OH, -SO2, =S, -COOH, -NR2, carbonyl, -C(O)-(C1-C 10alkyl)-CF3, -C(O)-CF3, -C(O)NR2, -(C1-C 10 Aryl)-S-(C6-C 10 aryl), -C(O)-(C6-C 10 aryl), -(CH2) m -O-(CH2) m -O-(C1~C 10 Examples of R groups include, but are not limited to, —C(O)NR, —C(S)NR, —SONR, —NRC(O)NR, —NRC(S)NR, salts thereof, and the like. Each R group in the foregoing list includes, but is not limited to, H, alkyl or substituted alkyl, aryl or substituted aryl, or alkaryl. When substituents are specified by a conventional chemical formula written from left to right, they equally encompass the chemically identical substituents that result from writing the structure from right to left (e.g., —CHO— is equivalent to —OCH—).

[0228] By way of example, substituents for alkyl and heteroalkyl radicals (including those groups referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) include, but are not limited to, -OR, ═O, ═NR, ═N—OR, —NR, —SR, -halogen, —SiR, —OC(O)R, —C(O)R, —COR, —CONR, —OC(O)NR, —NRC(O)R, —NRC(O)NR, —NR(O)R, —NR—C(NR)═NR, —S(O)R, —S(O)R, —S(O)NR, —NRSOR, —CN, and —NO. Each of the R groups in the above list includes, but is not limited to, hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl (including, but not limited to, aryl substituted with 1 to 3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or aralkyl groups. When two R groups are bonded to the same nitrogen atom, they can combine with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR2 is meant to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl.

[0229] By way of example, substituents for aryl and heteroaryl groups include, but are not limited to, -OR, =O, =NR, =N-OR, -NR, -SR, -halogen, -SiR, -OC(O)R, -C(O)R, -COR, -CONR, -OC(O)NR, -NRC(O)R, -NRC(O)NR, -NR(O)R, -NR-C(NR)=NR, -S(O)R, -S(O)R, -S(O)NR, -NRSOR, -CN, -NO, -R, -N, -CH(Ph), fluoro(C1-C4)alkoxy, and fluoro(C1-C4)alkyl, in a number ranging from 0 to the total number of open valences in the aromatic ring system, where each R group in the foregoing list includes, but is not limited to, hydrogen, alkyl, heteroalkyl, aryl, and heteroaryl.

[0230] As used herein, the term "therapeutically effective amount" refers to the amount of a composition comprising at least one non-natural amino acid polypeptide and / or at least one modified non-natural amino acid polypeptide administered to a patient already suffering from a disease, condition, or disorder to cure or at least partially arrest or alleviate some of the symptoms of the disease, condition, or disorder being treated. The effectiveness of such compositions will depend on the condition, including but not limited to, the severity and course of the disease, condition, or disorder, previous treatments, the patient's health status and response to drugs, and the judgment of the treating physician. By way of example, a therapeutically effective amount can be determined by routine experimentation, including but not limited to, a dose escalation clinical trial.

[0231] As used herein, the term "thioalkoxy" refers to a sulfur-containing alkyl group attached to the molecule via an oxygen atom.

[0232] The term "thermal melting temperature" or Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium.

[0233] As used herein, the term "toxic moiety" or "toxic group" refers to a compound that can cause damage, injury, or death. Toxic moieties include auristatins, DNA minor groove binders, DNA minor groove alkylators, enediynes, lexitropsins, duocarmycins, taxanes, puromycins, dolastatins, maytansinoids, vinca alkaloids, AFP, MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholinodoxorubicin, rhizoxin, cyanomorpholinodoxorubicin, dolastatin-10, echinomycin, and combretastatin. (combretatstatin), calicheamicin, maytansine, DM-1, netropsin, podophyllotoxin (e.g., etoposide, teniposide, etc.), bacactin and its derivatives, antitubulin substances, cryptophysin, combretastatin, auristatin E, vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, epothilone A, epothilone B, nocodazole, col ritin, colcimid, estramustine, cemadotin, discodermolide, maytansine, eleutherobin, mechlorethamine, cyclophosphamide, melphalan, carmustine, lomustine, semustine, streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide, itarabine, cytosine arabinoside, fluorouracil, floxiridine, 6-thioguanine, 6-mercaptopurine, pentostatin, 5-fluorouracil, methotrexate, 10-propargyl-5,8-dideazafolate, 5,8-dideazatetrahydrofolate, leucovorin, fludarabine phosphate, pentostatin, gemcitabine, Ara-C, paclitaxel, docetaxel, deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine, brequinar, antibiotics (e.g.,Anthracyclines, gentamicin, cephalothin, vancomycin, telavancin, daptomycin, azithromycin, erythromycin, rocithromycin, furazolidone, amoxicillin, ampicillin, carbenicillin, flucloxacillin, methicillin, penicillin, ciprofloxacin, moxifloxacin, ofloxacin, doxycycline, minocycline, oxytetracycline, tetracycline, streptomycin, rifabutin, ethambutol, rifaximin, etc.), antibiotics Antiviral drugs (e.g., abacavir, acyclovir, Ampligen, cidofovir, delavirdine, didanosine, efavirenz, entecavir, fosfonet, ganciclovir, ibacitabine, Immunovir, idoxuridine, inosine, lopinavir, methisazone, nexavir, nevirapine, oseltamivir, penciclovir, stavudine, trifluridine, Truvada, valacyclovir, zanamivir, etc.), daunorubicin hydrochloride, daunomycin, rubidomycin, cerubicin, idarubicin, doxorubicin, epirubicin, and morphine derivatives, phenoxyzolidinone cyclopeptides (e.g., dactinomycin), basic glycopeptides (e.g., bleomycin), anthraquinone glycosides (e.g., plicamycin, mithramycin), anthracenediones (e.g., mitoxantrone), azirinopyrroloindoledione (e.g., mitomycin), macrocyclic immunosuppressants (e.g., cyclosporine, FK-506, tacrolimus, prograf, rapamycin, etc.), navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxal "Taxanes" include, but are not limited to, cyclophosphamide, ifosamide, droloxafine, allocorchicine, halichondrin B, colchicine, colchicine derivatives, maytansine, rhizoxin, paclitaxel, paclitaxel derivatives, docetaxel, thiocolchicine, tritylcysterol, vinblastine sulfate, vincristine sulfate, cisplatin, carboplatin, hydroxyurea, N-methylhydrazine, epidophyllotoxin, procarbazine, mitoxantrone, leucovorin, and tegafur.In addition to any active taxane derivative or prodrug, paclitaxel is included.

[0234] As used herein, the terms "treat," "treating," or "treatment" include alleviating, relieving, or ameliorating the symptoms of a disease or condition, preventing additional symptoms, improving or preventing the underlying metabolic cause of a condition, suppressing a disease or condition, e.g., suppressing the progression of the development of a disease or condition, relieving a disease or condition, reversing a disease or condition, relieving abnormalities caused by a disease or condition, or arresting the symptoms of a disease or condition. The terms "treat," "treating," or "treatment" include, but are not limited to, prophylactic and / or therapeutic treatments.

[0235] As used herein, the term "water-soluble polymer" refers to any polymer that is soluble in aqueous solvents. Examples of such water-soluble polymers include polyethylene glycol, polyethylene glycol propionaldehyde, mono C1-C6 10 Alkoxy derivatives or mono C1-C 10These include, but are not limited to, aryloxy derivatives (as described in U.S. Pat. No. 5,252,714, which is incorporated herein by reference), monomethoxypolyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, divinyl ether maleic anhydride, N-(2-hydroxypropyl)-methacrylamide, dextran, dextran derivatives (including dextran sulfide), polypropylene glycol, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols, heparin, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives (including, but not limited to, methylcellulose and carboxymethylcellulose), serum albumin, starch and starch derivatives, polypeptides, polyalkylene glycols and derivatives thereof, copolymers of polyalkylene glycols and derivatives thereof, polyvinyl ethyl ether, and alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide], and the like, or mixtures thereof. By way of example, the conjugation of such water-soluble polymers to natural amino acid polypeptides or non-natural polypeptides results in alterations, including, but not limited to, improved aqueous solubility, extended or modified serum half-life, extended or modified therapeutic half-life compared to the unmodified form, improved bioavailability, modified biological activity, extended circulation time, modified immunogenicity, modified physical association properties (e.g., including, but not limited to, aggregation and multimerization), altered receptor binding, altered binding to one or more binding partners, and altered receptor dimerization or multimerization. Furthermore, the water-soluble polymer may or may not have its own biological activity.

[0236] Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art, are employed.

[0237] The compounds described herein (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides, modified non-natural amino acid polypeptides, and reagents for producing the aforementioned compounds) include isotopically labeled compounds. Isotopically labeled compounds are the same compounds depicted by the various formulas and structures described herein, except that one or more atoms have been replaced with an atom having an atomic mass or mass different from the atomic mass or mass found in nature. Examples of isotopes that may be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 2 H, 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 35 S, 18 F, 36 Certain isotopically labeled compounds described herein, such as compounds containing radioactive isotopes (e.g., 3 H and 14 Isotopically labeled compounds incorporating deuterium (i.e., C) are useful in drug and / or substrate tissue distribution assays. 2 Substitution with isotopes such as 3H may offer certain therapeutic advantages resulting from greater metabolic stability, such as, for example, extended in vivo half-life or reduced dosage requirements.

[0238] Some compounds described herein (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides, modified non-natural amino acid polypeptides, and reagents for producing the above-described compounds) possess asymmetric carbon atoms and can therefore exist as enantiomers or diastereomers. Diasteromeric mixtures can be separated into individual diastereomers based on their physicochemical differences, for example, by methods known as chromatography and / or fractal crystallization. Enantiomers can be resolved by converting the enantiomeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., alcohol), whereupon the diastereomers are resolved and the individual diastereomers are converted (e.g., by hydrolysis) into the corresponding pure enantiomers. All such isomers, including diastereomers, enantiomers, and mixtures thereof, are considered part of the compositions described herein.

[0239] In additional or further embodiments, the compounds described herein (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides, modified non-natural amino acid polypeptides, and reagents for producing the above-mentioned compounds) are used in the form of prodrugs. In additional or further embodiments, the compounds described herein (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides, modified non-natural amino acid polypeptides, and reagents for producing the above-mentioned compounds) are metabolized when administered to an organism in need of producing metabolic products, which are then utilized to produce a desired effect, including a desired therapeutic effect. In further or additional embodiments, the compounds are active metabolic products of the non-natural amino acids and "modified or unmodified" non-natural amino acid polypeptides.

[0240] The methods and formulations described herein include the use of N-oxides, crystalline forms (also known as polymorphs), or pharmaceutically acceptable salts of the non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides. In certain embodiments, the non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides can exist as tautomers. All tautomers are included within the scope of the non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides described herein. Furthermore, the non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides described herein can exist in unsolvated and solvated forms with pharmaceutically acceptable solvents (e.g., water, ethanol, etc.). Solvated forms of the non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides described herein are also considered to be described herein.

[0241] Some compounds described herein (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides, modified non-natural amino acid polypeptides, and reagents for producing the compounds described above) can exist in several tautomeric forms. All such tautomeric forms are considered part of the compositions described herein. Also, for example, all enol-keto forms of any compounds described herein (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides, modified non-natural amino acid polypeptides, and reagents for producing the compounds described above) are considered part of the compositions described herein.

[0242] Some compounds described herein (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides, modified non-natural amino acid polypeptides, and reagents for producing any of the foregoing compounds) are acidic and can form salts with pharmaceutically acceptable cations. Some compounds described herein (including, but not limited to, non-natural amino acid, non-natural amino acid polypeptides, modified non-natural amino acid polypeptides, and reagents for producing any of the foregoing compounds) are basic and can therefore form salts with pharmaceutically acceptable anions. All such salts, including disalts, are included within the scope of the compositions described herein and can be prepared by conventional methods. For example, salts can be prepared by contacting an acidic and a basic substance in an aqueous, non-aqueous, or partially aqueous medium. Salts are recovered using at least one of the following techniques: filtration, precipitation with a non-solvent followed by filtration, evaporation of the solvent, or, in the case of aqueous solutions, lyophilization.

[0243] Pharmaceutically acceptable salts of the non-natural amino acid polypeptides described herein can be generated when an acidic proton present in the parent non-natural amino acid polypeptide is replaced with a metal ion (e.g., an alkali metal ion, an alkaline earth ion, an aluminum ion, or an organic base coordinate). Additionally, salt forms of the non-natural amino acid polypeptides described herein can be prepared using salts of the starting materials or intermediates. The non-natural amino acid polypeptides described herein can be prepared as pharmaceutically acceptable acid addition salts (a type of pharmaceutically acceptable salt) by reacting the free base form of the non-natural amino acid polypeptides described herein with a pharmaceutically acceptable inorganic or organic acid. Alternatively, the non-natural amino acid polypeptides described herein can be prepared as pharmaceutically acceptable base addition salts (a type of pharmaceutically acceptable salt) by reacting the free acid form of the non-natural amino acid polypeptides described herein with a pharmaceutically acceptable inorganic or organic base.

[0244] Types of pharmaceutically acceptable salts include: (1) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or acid addition salts formed with acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 4-methylbicyclo-[2.2.2]oct-2-ene, and the like. (1) Acid addition salts formed with organic acids such as 4,4'-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, and muconic acid; and (2) salts formed when an acidic proton present in the parent compound is replaced by a metal ion (e.g., an alkali metal ion, an alkaline earth ion, an aluminum ion, or an organic base coordinate). Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, and N-methylglucamine. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like.

[0245] The corresponding counterions of pharmaceutically acceptable salts of non-natural amino acid polypeptides can be analyzed and identified using a variety of methods, including, but not limited to, ion exchange chromatography, ion chromatography, capillary electrophoresis, inductively coupled plasma, atomic absorption spectroscopy, mass spectrometry, or any combination thereof. Furthermore, the therapeutic activity of such pharmaceutically acceptable salts of non-natural amino acid polypeptides can be tested using the techniques and methods described in Examples 87-91.

[0246] Reference to a salt, of course, includes solvent addition forms or its crystalline forms, particularly solvates or polymorphs. Solvates contain stoichiometric or non-stoichiometric amounts of solvent and are often produced during the process of crystallization with pharmaceutically acceptable solvents such as water and ethanol. When the solvent is water, hydrates are produced, and when the solvent is alcohol, alcoholates are produced. Polymorphs include different crystal packing arrangements of the same component composition of a compound. Polymorphs usually have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardnesses, crystal shapes, optical and electrical properties, stability, and solubility. Various factors, such as the recrystallization solvent, crystallization rate, and storage temperature, result in a single dominant crystalline form.

[0247] Polymorphs and / or solvates of pharmaceutically acceptable salts of non-natural amino acid polypeptides can be screened and characterized using a variety of techniques, including, but not limited to, thermal analysis, X-ray diffraction, spectroscopy, vapor sorption, and microscopy. Thermal analysis methods involve thermochemical decomposition or thermophysical processing (including, but not limited to, polymorphic transitions), and are used to analyze polymorphic phase relationships, measure weight loss, detect glass transition temperatures, or conduct excipient compatibility studies. Such methods include, but are not limited to, differential scanning calorimetry (DSC), modulated differential scanning calorimetry (MDCS), thermogravimetric analysis (TGA), and thermogravimetric infrared analysis (TG / IR). X-ray diffraction methods include, but are not limited to, single crystal powder diffractometers and synchrotron radiation sources. Various spectroscopic techniques used include, but are not limited to, Raman, FTIR, UVIS, and NMR (liquid and solid state). Various microscopy techniques include, but are not limited to, polarized light microscopy, scanning electron microscopy (SEM) with energy dispersive X-ray analysis (EDX), environmental scanning electron microscopy with EDX (in a gas or water vapor atmosphere), IR microscopy, and Raman microscopy.

[0248] BRIEF DESCRIPTION OF THE DRAWINGS The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized. The accompanying drawings, in which:

[0249] FIG. 1 shows the binding of Her-tox to the Her2 receptor.

[0250] FIG. 2 shows the expression of Anti-Her2 mutants determined by ELISA analysis.

[0251] FIG. 3 shows the expression of Anti-Her2 mutants determined by ELISA analysis.

[0252] FIG. 4 shows a cell proliferation assay using the HCC1954 breast cancer cell line and dolastatin linker derivatives.

[0253] FIG. 5 shows the analysis of cell proliferation assays using HCC1954 breast cancer cell line and dolastatin linker derivatives.

[0254] FIG. 6 shows analysis of cell proliferation assays using the SKOV-3 ovarian cancer cell line and dolastatin linker derivatives.

[0255] FIG. 7 shows analysis of cell proliferation assays using the SKOV-3 ovarian cancer cell line and trastuzumab-tox conjugates.

[0256] FIG. 8 shows analysis of cell proliferation assays using MDA-MB-468 breast cancer cell line and dolastatin linker derivatives.

[0257] FIG. 9 shows analysis of cell proliferation assays using MDA-MB-468 breast cancer cell line and trastuzumab-tox conjugates.

[0258] [Figure 10] Measurement of tumor volume (unit: mm) after a single intracardiac administration of various trastuzumab-conjugated dolastatin derivatives (3.3 mg / kg, 10 mg / kg, 20 mg / kg). 3 ) is a diagram showing the same.

[0259] FIG. 11 shows the assay format used to measure the concentrations of various trastuzumab-conjugated dolastatin derivatives in SD rat serum.

[0260] [Figure 12] A graph showing the serum concentrations (unit: ng / mL) of various trastuzumab-conjugated dolastatin derivatives after a single intravenous injection.

[0261] Figure 13 shows serum concentrations (in ng / mL) of various trastuzumab-conjugated dolastatin derivatives after a single intravenous injection. This assay detects antibody binding to the ErbB2 receptor.

[0262] Figure 14 shows the serum concentrations (unit: ng / mL) of various trastuzumab-conjugated dolastatin derivatives after a single intravenous injection. In vivo stability measurements detect at least two dolastatin derivatives conjugated to trastuzumab.

[0263] FIG. 15 shows changes in body weight and tumor volume in rats treated with various trastuzumab-conjugated dolastatin derivatives.

[0264] Figure 16 shows the antitumor effects of trastuzumab, Her2-HS122-NCD1, and Her2-HS122 / LK145-HCD1 against established HCC1954 tumors in SCID-bg mic mice. Mice received a single intravenous injection on day 1 (indicated by arrow). Data points represent group means of tumor volume, and error bars represent standard error.

[0265] FIG. 17 shows the antitumor effects of various dolastatin linker derivatives in an MDA361DYT2 breast (2+) xenograft model.

[0266] FIG. 18 shows the antitumor effects of various dolastatin linker derivatives in an MDA361DYT2 breast (2+) xenograft model.

[0267] Detailed Description of the Invention While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Numerous changes, modifications, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0268] (I. Introduction) In recent years, an entirely new technology in protein science has been reported that overcomes many of the limitations associated with site-specific modification of proteins. Specifically, new components have been added to the protein biosynthetic machinery of the prokaryotic Escherichia coli (E. coli) (e.g., L. Wang, et al., (2001), Science 292:498-500) and the eukaryotic yeast Sacchromyces cerevisiae (S. cerevisiae) (e.g., J. Chin et al., Science 301:964-7 (2003)). These components enable the incorporation of non-genetically encoded amino acids into proteins in vivo. Using this methodology, many new amino acids with novel chemical, physical, or biological properties (including photoaffinity-labeled amino acids, photoisomerizable amino acids, ketoamino acids, and glycosylated amino acids) have been efficiently and with high fidelity incorporated into proteins in E. coli and yeast in response to the amber codon TAG. For example, JW Chin et al., (2002), Journal of the American Chemical Society 124:9026-9027 (incorporated by reference); JW Chin, & P. G. Schultz, (2002), ChemBioChem 3(11):1135-1137 (incorporated by reference); JW Chin, et al., (2002), PNAS United States of America 99(17):11020-11024 (incorporated by reference in its entirety); and, L. Wang, & P.G. Schultz, (2002), Chem. Comm., 1-11 (incorporated by reference). These studies demonstrate that it is possible to selectively and routinely introduce chemical functional groups not found in proteins that are chemically inert toward all of the functional groups found on the 20 common genetically encoded amino acids and can be used to react efficiently and selectively to form stable covalent bonds.

[0269] (II. Overview) At one level, described herein are tools (methods, compositions, techniques) for making and using dolastatin linker derivatives or analogs that include at least one carbonyl, dicarbonyl, oxime, hydroxylamine, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, azide, amidine, imine, diamine, ketoamine, ketoalkyne, alkyne, cycloalkyne, or enedione. At another level, described herein are tools (methods, compositions, techniques) for making and using dolastatin linker derivatives or analogs that include at least one unnatural amino acid or an unnatural amino acid modified with an oxime, aromatic amine, heterocycle (e.g., indole, quinoxaline, phenazine, pyrazole, triazole, etc.).

[0270] Such dolastatin linker derivatives containing unnatural amino acids may contain additional functionalities. Functionalities include, but are not limited to, polymers, water-soluble polymers, polyethylene glycol derivatives, second proteins, polypeptides or polypeptide analogs, antibodies or antibody fragments, and any combination thereof. Note that the various functionalities described above are not intended to imply that a member of one functionality cannot be classified as a member of another functionality. In fact, overlap exists depending on the specific situation. As just one example, water-soluble polymers overlap in scope with polyethylene glycol derivatives, but this overlap is not complete, and therefore both functionalities are listed above.

[0271] In some embodiments, described herein are toxic group linker derivatives that include a carbonyl, dicarbonyl, oxime, hydroxylamine, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, azide, amidine, imine, diamine, ketoamine, ketoalkyne, alkyne, cycloalkyne, or enedione. In some embodiments, the toxic group derivative comprises any of the linkers disclosed herein. In other embodiments, described herein are tools (methods, compositions, techniques) for making and using toxic group derivatives or analogs that include at least one unnatural amino acid or an unnatural amino acid modified with an oxime, aromatic amine, or heterocycle (e.g., indole, quinoxaline, phenazine, pyrazole, triazole, etc.).

[0272] In some embodiments, such toxic group derivatives containing unnatural amino acids may contain additional functionality. Functionalities include, but are not limited to, polymers, water-soluble polymers, derivatives of polyethylene glycol, second proteins, polypeptides or polypeptide analogs, antibodies or antibody fragments, and any combination thereof. In certain embodiments, the toxic group derivative is a dolastatin or an auristatin. In certain embodiments, the toxic group derivative is dolastatin-10. Note that the various functionalities listed above are not intended to suggest that members of one functionality cannot be classified as members of another functionality. In fact, overlap exists in certain situations. As just one example, water-soluble polymers overlap in scope with derivatives of polyethylene glycol, but this overlap is not complete, and therefore both functionalities are listed above.

[0273] In some embodiments, the present invention describes the preparation of certain toxic moieties with linkers that reduce the toxicity of the moiety in vivo while maintaining pharmacological activity of the toxic moiety. In some embodiments, the toxicity of the attached toxic group is reduced or eliminated when administered to animals or humans compared to the free toxic group or toxic group derivative containing a labile linkage, while maintaining pharmacological activity. In some embodiments, increased doses of the attached toxic group (e.g., dolastatin linker derivatives, non-natural amino acid attached dolastatin derivatives) can be administered to animals or humans with high safety. In some embodiments, non-natural amino acid polypeptides (e.g., dolastatin derivatives) attached to a toxic moiety provide stability in vivo and in vitro. In some embodiments, non-natural amino acid polypeptides (e.g., dolastatin-10 derivatives) attached to a toxic moiety are effective and have reduced toxicity compared to the free toxic group (e.g., dolastatin-10).

[0274] III. Dolastatin Linker Derivatives At one level, described herein are tools (methods, compositions, and techniques) for making and using dolastatin linker derivatives or analogs containing at least one unnatural amino acid or an unnatural amino acid modified with a carbonyl, dicarbonyl, oxime, or hydroxylamine group. Such dolastatin linker derivatives containing unnatural amino acids may contain additional functionality. Functionality includes, but is not limited to, polymers, water-soluble polymers, polyethylene glycol derivatives, polypeptides or polypeptide analogs, second proteins, antibodies or antibody fragments, and any combination thereof. It should be noted that the various functionalities described above are not intended to suggest that a member of one functionality cannot be classified as a member of another functionality. Indeed, overlap exists in certain circumstances. As just one example, water-soluble polymers overlap in scope with polyethylene glycol derivatives, but this overlap is not complete, and therefore both functionalities are listed above.

[0275] In certain embodiments, methods for selecting and designing dolastatin linker derivatives to be modified using these methods, compositions, and techniques are described herein. Novel dolastatin linker derivatives may be designed de novo, by way of example only, as part of a high-throughput screening method (in which a large number of polypeptides may be designed, synthesized, characterized, and / or tested) or based on a researcher's interest. Novel dolastatin linker derivatives may also be designed based on known or partially characterized polypeptides. By way of example only, dolastatins have been the subject of significant research in the scientific community, and novel compounds may be designed based on the dolastatin structure. Principles for selecting which amino acids to replace and / or modify are described herein, respectively. Selection of which modifications to employ is also described herein and can be tailored to the needs of the experimenter or end user. Such needs include, but are not limited to, manipulating the therapeutic effect of the polypeptide, improving the safety profile of the polypeptide, and adjusting the pharmacokinetics, pharmacology, and / or potency of the polypeptide, including, by way of example only, improving water solubility, improving bioavailability, increasing serum half-life, increasing therapeutic half-life, modulating immunogenicity, modulating biological activity, or extending circulation time. Additionally, such modifications include, by way of example only, providing additional functionality to the polypeptide, incorporating antibodies, and any combination of the foregoing modifications.

[0276] Also described herein are dolastatin linker derivatives that have, or can be modified to contain, oxime, carbonyl, or hydroxylamine groups, including methods for making, purifying, characterizing, and using such dolastatin linker derivatives.

[0277] The dolastatin linker derivative may contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or 10 or more carbonyl or dicarbonyl groups, oxime groups, hydroxylamine groups, or protected forms thereof. The dolastatin linker derivatives may be the same or different. For example, the derivative may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more different sites containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more different reactive groups.

[0278] A. Structure and Synthesis of Dolastatin Linker Derivatives: Electrophilic and Nucleophilic Groups Dolastatin derivatives containing a hydroxylamine group (also called aminooxy) in the linker can react with various electrophilic groups to form conjugates (including, but not limited to, PEG and other water-soluble polymers). The enhanced nucleophilicity of the aminooxy group, such as hydrazine, hydrazide, and semicarbazide, allows it to react efficiently and selectively with a variety of molecules containing carbonyl or dicarbonyl groups. These carbonyl or dicarbonyl groups include, but are not limited to, ketones, aldehydes, or other functional groups with similar chemical reactivity. See, for example, Shao, J. and Tam, J., J. Am. Chem. Soc. 117:3893-3899 (1995); H. Hang and C. Bertozzi, Acc. Chem. Res. 34(9): 727-736 (2001). Reaction with a hydrazine group results in the corresponding hydrazone, while oximes are generally obtained by reacting an aminooxy group with a carbonyl or dicarbonyl group, including, but not limited to, a ketone, aldehyde, or other functional group with similar chemical reactivity. In some embodiments, dolastatin derivatives containing an azide, alkyne, or cycloalkyne linker allow for the attachment of molecules via cycloaddition reactions (e.g., 1,3-dipolar cycloaddition or azide-alkyne Huisgen cycloaddition) (as described in U.S. Patent 7,807,619, the scope of which is incorporated herein by reference).

[0279] Thus, in certain embodiments, dolastatin derivatives are described herein that include a linker containing a hydroxylamine, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, ketoamine, ketoalkyne, or enedione hydroxylamine group, a hydroxylamine-like group (similar in reactivity to and structurally similar to a hydroxylamine group), a masked hydroxylamine group (readily convertible to a hydroxylamine group), or a protected hydroxylamine group (similar in reactivity to a hydroxylamine group upon deprotection). Such dolastatin linker derivatives include compounds having the structures of Formulae (I), (III), (IV), (V), and (VI).

[0280] [ka] JPEG0007638942000058.jpg193169

[0281] (In the formula, Z has the following structure:

[0282] [ka]

[0283] R5 is H, COR8, alkyl having 1 to 6 carbon atoms, or thiazole; R8 is OH or -NH-(alkylene-O) n -NH2, R6 is OH or H; Ar is phenyl or pyridine; R7 is alkyl having 1 to 6 carbon atoms or hydrogen; Y and V are each selected from the group consisting of hydroxylamine, methyl, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, azide, ketoamine, ketoalkyne, alkyne, cycloalkyne, and enedione; L, L1, L2, L3, and L4 are each a bond, -alkylene-, -alkylene-C(O)-, -alkylene-J-, or -(alkylene-O)-, respectively. n -Alkylene-, -(alkylene-O) n -Alkylene-C(O)-, -(Alkylene-O) n -J-, -(alkylene-O) n -J-alkylene, -(alkylene-O) n -(CH2) n’ -NHC(O)-(CH2) n’’ -C(Me)2-SS-(CH2) n’’’ -NHC(O)-(alkylene-O) n’’’’ -Alkylene, -(alkylene-O) n -Alkylene-W-, -Alkylene-C(O)-W-, -(Alkylene-O) n -Alkylene-J-, -Alkylene'-J-(Alkylene-O) n -Alkylene-, -(alkylene-O) n -Alkylene-J-alkylene'-, -J-(alkylene-O) n -Alkylene-, -(alkylene-O) n -Alkylene-J-(Alkylene-O) n '-Alkylene-J'-, -W-, -Alkylene-W-, Alkylene'-J- (Alkylene-NMe) n -Alkylene-W-, -J-(Alkylene-NMe) n -Alkylene-W-, -(Alkylene-O) n -Alkylene-UC(O)-, -(Alkylene-O) n-alkylene-U-alkylene; -J-alkylene-NMe-alkylene'-NMe-alkylene''-W-, and -alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene'''-W-; W has the following structure:

[0284] [ka]

[0285] U has the structure:

[0286] [ka]

[0287] Each J and J' independently has the structure:

[0288] [ka]

[0289] n, n', n'', n''', and n'''' are each independently an integer of 1 or greater; Or, L is absent, Y is methyl, R5 is COR8, and R8 is -NH(alkylene-O) n -NH2.) Such dolastatin linker derivatives may take the form of a salt or may be incorporated into unnatural amino acids, polymers, polysaccharides, or polynucleotides, optionally post-translationally modified.

[0290] In some embodiments of compounds of Formula (I), (III), and (V), R5 is thiazole or carboxylic acid. In some embodiments of compounds of Formula (I), (III), and (V), R5 is hydrogen. In some embodiments of compounds of Formula (I), (III), and (V), R5 is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or hexyl. In some embodiments of compounds of Formula (I), (III), and (V), R5 is -NH-(alkylene-O) n and the alkylene is -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, or -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-. In certain embodiments of compounds of Formula (IV) and (VI), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 , 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

[0291] In some embodiments, Y is azide. In other embodiments, Y is a cycloalkyne. In particular embodiments, the cycloalkyne has the structure:

[0292] [ka]

[0293] (In the formula, Each R 19 are independently selected from the group consisting of alkyl having 1 to 6 carbon atoms, alkoxy having 1 to 6 carbon atoms, ester, ether, thioether, aminoalkyl, halogen, alkyl ester, aryl ester, amide, aryl amide, alkyl halide, alkylamine, alkylsulfonic acid, alkylnitro, thioester, sulfonyl ester, halosulfonyl, nitrile, alkylnitrile, and nitro; q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.) In certain embodiments of compounds of Formula (I), (III), and (V), R6 is hydrogen. In some embodiments of compounds of Formula (I), (III), and (V), R6 is hydroxy.

[0294] In certain embodiments of compounds of Formula (I), (III), and (V), Ar is phenyl.

[0295] In some embodiments of compounds of Formula (I), (III), (IV), (V), and (V), R7 is methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, pentyl, or hexyl. In some embodiments of compounds of Formula (I), (III), (IV), (V), and (V), R7 is hydrogen.

[0296] In certain embodiments of compounds of Formula (I), (III), and (V), Y is a hydroxylamine, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, ketoamine, ketoalkyne, alkyne, or enedione.

[0297] In certain embodiments of compounds of Formula (IV) and (VI), V is hydroxylamine, methyl, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, ketoamine, ketoalkyne, and enedione.

[0298] In some embodiments of compounds of Formula (I), (III), (IV), (V), and (VI), L, L1, L2, L3, and L4 are each independently a cleavable or non-cleavable linker. In some embodiments of compounds of Formula (I), (III), (IV), (V), and (VI), L, L1, L2, L3, and L4 are each independently an oligo(ethylene glycol)-derivatized linker.

[0299] In certain embodiments of compounds of Formula (I), (III), (IV), (V), and (VI), the alkylene, alkylene', alkylene'', and alkylene''' are each independently -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2C H2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH 2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, or -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-. In certain embodiments of compounds of Formula (XIV), (XV), (XVI), (XVII), and (XVIII), n, n', n'', n''', and n''' are 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 0, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

[0300] B. Structure and Synthesis of Dolastatin Linker Derivatives: Hydroxylamine Group Thus, in certain embodiments, dolastatin derivatives are described herein that include a hydroxylamine group, a hydroxylamine-like group (which has similar reactivity to and is structurally similar to a hydroxylamine group), a masked hydroxylamine group (which can be readily converted to a hydroxylamine group), or a protected hydroxylamine group (which has similar reactivity to a hydroxylamine group upon deprotection) in the linker. Such dolastatin linker derivatives include compounds having the structure of Formula (I):

[0301] [ka]

[0302] (In the formula, Z has the following structure:

[0303] [ka]

[0304] R5 is H, COR8, alkyl having 1 to 6 carbon atoms, or thiazole; R8 is OH or -NH-(alkylene-O) n -NH2, R6 is OH or H; Ar is phenyl or pyridine; R7 is alkyl having 1 to 6 carbon atoms or hydrogen; Y is NH2-O- or methyl; L is -alkylene-, -alkylene-C(O)-, -(alkylene-O) n -Alkylene-, -(alkylene-O) n -Alkylene-C(O)-, -(Alkylene-O) n -(CH2) n’ -NHC(O)-(CH2) n’’ -C(Me)2-SS-(CH2) n’’’ -NHC(O)-(alkylene-O) n’’’’-Alkylene, -(alkylene-O) n -Alkylene-W-, -Alkylene-C(O)-W-, -(Alkylene-O) n -alkylene-U-alkylene-C(O)-, and -(alkylene-O) n -alkylene-U-alkylene; W has the following structure:

[0305] [ka]

[0306] U has the structure:

[0307] [ka]

[0308] Or, L is absent, Y is methyl, R5 is COR8, and R8 is -NH(alkylene-O) n -NH2, and n, n', n'', n''', and n'''' are each independently an integer of 1 or greater. Such dolastatin linker derivatives may take the form of a salt or may be incorporated into unnatural amino acids, polymers, polysaccharides, or polynucleotides, optionally post-translationally modified.

[0309] In some embodiments of a compound of Formula (I), R5 is thiazole. In some embodiments of a compound of Formula (I), R5 is hydrogen. In some embodiments of a compound of Formula (I), R5 is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or hexyl. In some embodiments of a compound of Formula (I), R5 is -NH-(alkylene-O) nand the alkylene is -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, or -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-. In certain embodiments of a compound of Formula (I), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

[0310] In certain embodiments of the compound of Formula (I), R6 is hydrogen. In some embodiments of the compound of Formula (I), R6 is hydroxy.

[0311] In certain embodiments of a compound of Formula (I), Ar is phenyl.

[0312] In some embodiments of a compound of Formula (I), R7 is methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, pentyl, or hexyl. In some embodiments of a compound of Formula (I), R7 is hydrogen.

[0313] In certain embodiments of a compound of Formula (I), Y is hydroxylamine, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, ketoamine, ketoalkyne, or enedione. In certain embodiments of a compound of Formula (I), V is hydroxylamine, methyl, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, ketoamine, ketoalkyne, and enedione.

[0314] In some embodiments of a compound of Formula (I), each L is independently a cleavable linker or a non-cleavable linker. In some embodiments of a compound of Formula (I), each L is independently an oligo(ethylene glycol)-derivatized linker.

[0315] In certain embodiments of a compound of Formula (I), each alkylene is -CH-, -CHCH-, -CHCHCH-, -CHCHCHCH-, -CHCHCHCHCH-, -CHCHCHCHCHCH-, -CHCHCHCHCH2CH2CH2-, -CHCH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, or -CHCH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-. In certain embodiments of a compound of Formula (I), n, n', n'', n''', and n'''' are each 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 , 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

[0316] In certain embodiments, the dolastatin linker derivative comprises a compound having the structure of Formula (II):

[0317] [ka]

[0318] In some embodiments of the compound of Formula (II), L is -(alkylene-O) nIn some embodiments of compounds of Formula (II), L is -(alkylene-). In some embodiments, each alkylene is -CHCH-, n=3, and R7 is methyl. In some embodiments of compounds of Formula (II), L is -(alkylene-O) n In some embodiments of the compound of Formula (II), L is -(alkylene-C(O)-. In some embodiments of the compound of Formula (II), each alkene is -CHCH-, n=4, and R is methyl. In some embodiments of the compound of Formula (II), L is -(alkylene-O) n -(CH2) n’ -NHC(O)-(CH2) n’’ -C(Me)2-SS-(CH2) n’’’ -NHC(O)-(alkylene-O) n’’’’ In some embodiments of compounds of formula (II), each alkene is -CHCH-, n=1, n'=2, n''=1, n'''=2, and n''''=4, and R7 is methyl. Such dolastatin linker derivatives may take the form of a salt, be incorporated into unnatural amino acids, polymers, polysaccharides, or polynucleotides, and optionally be post-translationally modified.

[0319] In some embodiments of compounds of Formula (II), each L is independently a cleavable linker or a non-cleavable linker. In some embodiments of compounds of Formula (II), each L is independently an oligo(ethylene glycol)-derivatized linker.

[0320] In some embodiments of a compound of Formula (II), R7 is methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, pentyl, or hexyl. In some embodiments of a compound of Formula (II), R7 is hydrogen.

[0321] In certain embodiments of a compound of Formula (II), each alkylene is -CH-, -CHCH-, -CHCHCH-, -CHCHCHCH-, -CHCHCHCHCH-, -CHCHCHCHCHCH-, -CHCHCHCHCH2CH2CH2-, -CHCH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, or -CHCH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-. In certain embodiments of a compound of Formula (II), n, n', n'', n''', and n''' are 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 5, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

[0322] Such dolastatin linker derivatives include compounds having the structure of formula (III), (IV), (V), or (VI).

[0323] [ka] JPEG0007638942000070.jpg169169 JPEG0007638942000071.jpg108169

[0324] (In the formula, Z has the following structure:

[0325] [ka]

[0326] R5 is H, COR8, alkyl having 1 to 6 carbon atoms, or thiazole; R8 is OH; R6 is OH or H; Ar is phenyl or pyridine; R7 is alkyl having 1 to 6 carbon atoms or hydrogen; Y is NH2-O-, V is O-NH2, L1, L2, L3, and L4 each independently represent a bond, -alkylene-, or -(alkylene-O) n -Alkylene-J-, -Alkylene'-J-(Alkylene-O) n -Alkylene-, -J-(Alkylene-O) n -Alkylene-, -(Alkylene-O) n -Alkylene-J-(Alkylene-O) n '-Alkylene-J'-, -(Alkylene-O) n -Alkylene-J-alkylene'-, -W-, -Alkylene-W-, Alkylene'-J-(Alkylene-NMe) n -Alkylene-W-, -J-(Alkylene-NMe) n -alkylene-W-, -J-alkylene-NMe-alkylene'-NMe-alkylene''-W-, and -alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene''''-W-; W has the following structure:

[0327] [ka]

[0328] Each J and J' independently has the structure:

[0329] [ka]

[0330] (Each n and n' is independently an integer greater than or equal to 1.) Such dolastatin linker derivatives may be in the form of a salt, incorporated into unnatural amino acids, polymers, polysaccharides, or polynucleotides, and optionally post-translationally modified.

[0331] In some embodiments of a compound of Formula (III), (IV), (V), or (VI), R5 is thiazole. In some embodiments of a compound of Formula (III), (IV), (V), or (VI), R5 is hydrogen. In some embodiments of a compound of Formula (III), (IV), (V), or (VI), Ar is phenyl. In some embodiments of a compound of Formula (III), (IV), (V), or (VI), R7 is methyl. In some embodiments of a compound of Formula (III), (IV), (V), or (VI), n and n' are integers from 0 to 20. In some embodiments of a compound of Formula (III), (IV), (V), or (VI), n and n' are integers from 0 to 10. In some embodiments of a compound of Formula (III), (IV), (V), or (VI), n and n' are integers from 0 to 5.

[0332] In some embodiments of compounds of Formula (III) and (V), R5 is thiazole or carboxylic acid. In some embodiments of compounds of Formula (III) and (V), R5 is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or hexyl. In some embodiments of compounds of Formula (III) and (V), R5 is -NH-(alkylene-O) nand the alkylene is -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, or -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-. In certain embodiments of a compound of Formula (I), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

[0333] In certain embodiments of compounds of Formula (III), (IV), (V), and (VI), R6 is hydrogen. In some embodiments of compounds of Formula (III), (IV), (V), and (VI), R6 is hydroxy.

[0334] In certain embodiments of compounds of Formula (III), (IV), (V), and (VI), Ar is phenyl.

[0335] In some embodiments of compounds of Formula (III), (IV), (V), and (VI), R7 is methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, pentyl, or hexyl. In some embodiments of compounds of Formula (III), (IV), (V), and (VI), R7 is hydrogen.

[0336] In some embodiments of compounds of Formula (III) and (V), Y is hydroxylamine, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, ketoamine, ketoalkyne, or enedione. In some embodiments of compounds of Formula (IV) and (VI), V is hydroxylamine, methyl, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, ketoamine, ketoalkyne, or enedione.

[0337] In some embodiments of compounds of Formula (XIV), (XV), (XVI), (XVII), and (XVII), L1, L2, L3, and L4 are each independently a cleavable linker or a non-cleavable linker. In some embodiments of compounds of Formula (XIV), (XV), (XVI), (XVII), and (XVIII), L1, L2, L3, and L4 are each independently an oligo(ethylene glycol)-derivatized linker.

[0338] In certain embodiments of compounds of Formula (III), (IV), (V), and (VI), each alkylene is -CH-, -CHCH-, -CHCHCH-, -CHCHCHCH-, -CHCHCHCHCH-, -CHCHCHCHCHCH-, -CHCHCHCHCHCHCH-, -CHCH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, or -CHCH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-. In certain embodiments of compounds of Formula (III), (IV), (V), and (VI), said alkylene is methylene, ethylene, propylene, butylene, pentylene, hexylene, or heptylene.

[0339] In certain embodiments of compounds of Formula (III), (IV), (V), and (VI), n, n′, and n′ are each 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 , 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

[0340] In certain embodiments, the dolastatin linker derivative comprises a compound having the structure of Formula (II):

[0341] [ka]

[0342] In certain embodiments of the compound of Formula (VII), L is -(alkylene-O) n -alkylene-J-, and L2 is -alkylene'-J'-(alkylene-O) n '-Alkylene-, L3 is -J''-(Alkylene-O) n -alkylene-, alkylene is -CHCH-, alkylene is -(CH)-, n is 1, and n' and n'' are 3; J has the following structure:

[0343] [ka]

[0344] J' and J'' have the following structure:

[0345] [ka]

[0346] R7 is methyl. In certain embodiments of a compound of Formula (VII), L1 is -J-(alkylene-O) n -alkylene-, and L2 is -(alkylene-O) n' -alkylene-J'-alkylene'-, and L3 is -(alkylene-O) n'' -alkylene-J″-, where alkylene is —CH2CH2—, alkylene′ is —(CH2)4—, n is 1, n′ and n″ are 4, and J, J′, and J″ have the following structure:

[0347] [ka]

[0348] Such dolastatin linker derivatives may take the form of a salt or may be incorporated into unnatural amino acids, polymers, polysaccharides, or polynucleotides, optionally post-translationally modified.

[0349] In one embodiment, the compounds of Formulae (I) to (VII) are stable in aqueous solution under weakly acidic conditions for at least one month. In one embodiment, the compounds of Formulae (I) to (VII) are stable in aqueous solution under weakly acidic conditions for at least two weeks. In one embodiment, the compounds of Formulae (I) to (VII) are stable in aqueous solution under weakly acidic conditions for at least five days. In one embodiment, the weakly acidic conditions are pH 2 to 8.

[0350] The methods and compositions provided and described herein include polypeptides comprising dolastatin linker derivatives containing at least one carbonyl or dicarbonyl group, oxime group, hydroxylamine group, or protected or masked forms thereof. The introduction of at least one reactive group into a dolastatin linker derivative allows for the application of conjugation chemistries that do not react with common amino acids but participate in specific chemical reactions with one or more dolastatin linker derivatives, including, but not limited to, those described herein. Once incorporated, the dolastatin linker derivative side chains can be modified using chemical methodologies described herein or appropriate for the specific functional groups or substituents present on the dolastatin linker derivative.

[0351] The dolastatin linker derivative methods and compositions described herein provide conjugates of substances having various functional groups, substituents, or moieties with other substances, including, but not limited to, polymers, water-soluble polymers, derivatives of polyethylene glycol, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, and any combination thereof.

[0352] In certain embodiments, the dolastatin linker derivatives, linkers, and reagents described herein, including compounds of Formulae (I)-(VII), are stable in aqueous solution under weakly acidic conditions (including, but not limited to, pH 2-8). In other embodiments, such compounds are stable in aqueous solution under weakly acidic conditions for at least one month. In certain embodiments, such compounds are stable in aqueous solution under weakly acidic conditions for at least two weeks. In certain embodiments, such compounds are stable in aqueous solution under weakly acidic conditions for at least five days.

[0353] Another aspect of the compositions, methods, techniques, and strategies described herein is a method for studying or using any of the "modified or unmodified" unnatural amino acid dolastatin linker derivatives described above. This aspect includes, by way of example only, therapeutic, diagnostic, assay-based, industrial, cosmetic, plant biology, environmental, energy production, consumer product, and / or military uses that would benefit from dolastatin linker derivatives, including "modified or unmodified" unnatural amino acid polypeptides or proteins.

[0354] Dolastatin linker derivatives include, but are not limited to:

[0355] [ka] JPEG0007638942000080.jpg225169 JPEG0007638942000081.jpg212169 JPEG0007638942000082.jpg214169 JPEG0007638942000083.jpg216169 JPEG0007638942000084.jpg159169

[0356] IV. Unnatural Amino Acid DerivativesThe unnatural amino acids used in the methods and compositions described herein have at least one of the following four properties: (1) at least one functional group in the side chain of the unnatural amino acid has at least one property and / or activity and / or reactivity that is orthogonal to the chemical reactivity of the 20 common genetically encoded amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) or at least orthogonal to the chemical reactivity of a naturally occurring amino acid present in a polypeptide that includes the unnatural amino acid; (2) the introduced unnatural amino acid is substantially chemically inert relative to the 20 common genetically encoded amino acids; and (3) the unnatural amino acid preferably has stability comparable to the naturally occurring amino acids or can be stably incorporated into polypeptides under typical physiological conditions. More preferably, such incorporation can occur via an in vivo system. (4) The unnatural amino acid contains an oxime functional group or, preferably, a functional group that can be converted to an oxime group by reaction with a reagent under conditions that do not destroy the biological properties of the polypeptide containing the unnatural amino acid (unless, of course, such destruction of biological properties is the goal of the modification / transformation), or under conditions that allow the transformation to occur under aqueous conditions at a pH between about 4 and about 8, or under conditions where the reactive moiety on the unnatural amino acid is an electrophilic moiety. Any number of unnatural amino acids can be introduced into the polypeptide. The unnatural amino acid can further contain a protected or masked oxime, or a protected or masked group that can be converted to an oxime group after deprotection or unmasking.The unnatural amino acid may further comprise a protected or masked carbonyl or dicarbonyl group that can be transformed into a carbonyl or dicarbonyl group after deprotection or unmasking, thereby reacting with a hydroxylamine or an oxime to form an oxime group.

[0357] Unnatural amino acids that can be used in the methods and compositions described herein include, but are not limited to, amino acids with novel functional groups, amino acids that interact covalently or non-covalently with other molecules, glycosylated amino acids (e.g., sugar-substituted serines), amino acids modified with other carbohydrates, keto-containing amino acids, aldehyde-containing amino acids, amino acids containing polyethylene glycol or other polyethers, amino acids substituted with heavy elements, chemically cleavable and / or photocleavable amino acids, amino acids with extended side chains relative to natural amino acids, including, but not limited to, polyethers or long hydrocarbon chains (including, but not limited to, greater than about 5 or about 10 carbons), amino acids containing carbon-linked sugars, redox-active amino acids, aminothioacid-containing amino acids, and amino acids containing one or more toxic moieties.

[0358] In some embodiments, the unnatural amino acid comprises a glycan moiety. Examples of such amino acids include, but are not limited to, N-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine, N-acetyl-L-glucosaminyl-L-threonine, N-acetyl-L-glucosaminyl-L-asparagine, and O-mannosaminyl-L-serine. Examples of such amino acids also include those in which the naturally occurring N- or O-linkage between the amino acid and the glycan is replaced with a covalent bond not normally found in nature, including, but not limited to, an alkene, oxime, thioether, and amide. Examples of such amino acids also include glycans not normally found in naturally occurring proteins, such as 2-deoxyglucose and 2-deoxygalactose.

[0359] Chemical moieties incorporated into polypeptides through the incorporation of unnatural amino acids into the polypeptides offer various advantages and manipulations of the polypeptides. For example, the inherent reactivity of carbonyl or dicarbonyl functional groups (such as keto or aldehyde functional groups) allows for the selective modification of proteins in vivo and in vitro using any of a number of hydrazine- or hydroxylamine-containing reagents. Heavy atom unnatural amino acids can be useful, for example, for phasing X-ray structural data. Site-specific introduction of heavy atoms using unnatural amino acids also provides selectivity and flexibility in the selection of positions for the heavy atoms. Photoreactive unnatural amino acids (including, but not limited to, amino acids with benzophenone and aryl azide (including, but not limited to, phenyl azide) side chains) enable, for example, efficient photocrosslinking of polypeptides in vivo and in vitro. Examples of photoreactive amino acids include, but are not limited to, p-azido-phenylalanine and p-benzoyl-phenylalanine. Polypeptides bearing photoreactive amino acids can consequently be photocrosslinked at will by excitation of a temporal control that supplies the photoreactive group. In one non-limiting example, the methyl group of an unnatural amino acid can be replaced with a radiolabeled methyl group (including but not limited to) as a probe of local structure and dynamics, including but not limited to, with the use of nuclear magnetic resonance and vibrational microscopy.

[0360] A. Structure and Synthesis of Unnatural Amino Acid Derivatives: Carbonyl, Carbonyl-Like, Masked, and Protected Carbonyl Groups Amino acids having electrophilic reactive groups are capable of various reactions and can bond molecules via various chemical reactions, including, but not limited to, nucleophilic addition reactions. Examples of such electrophilic reactive groups include carbonyl or dicarbonyl groups (including keto or aldehyde groups), carbonyl- or dicarbonyl-like groups (which have similar reactivity to and are structurally similar to carbonyl or dicarbonyl groups), masked carbonyl or dicarbonyl groups (which can be readily converted to carbonyl or dicarbonyl groups), or protected carbonyl or dicarbonyl groups (which, when deprotected, have similar reactivity to carbonyl or dicarbonyl groups). Examples of such amino acids include amino acids having the structure of formula (XXXVII):

[0361] [ka]

[0362] (In the formula, A is optionally, if present, lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (where k is 1, 2, or 3), -S(O) k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; K is

[0363] [ka]

[0364] and R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; each R" is independently H, alkyl, substituted alkyl, or a protecting group, or when more than one R" group is present, two R" groups optionally form a heterocycloalkyl; R1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R3 and R4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4, or two R3 groups, may optionally form a cycloalkyl or heterocycloalkyl, or -ABKR groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl containing at least one carbonyl group (including a dicarbonyl group), protected carbonyl group (including a protected dicarbonyl group), or masked carbonyl group (including a masked dicarbonyl group); or the -KR groups together form a monocyclic or bicyclic cycloalkyl or heterocycloalkyl containing at least one carbonyl group (including a dicarbonyl group), protected carbonyl group (including a protected dicarbonyl group), or masked carbonyl group (including a masked dicarbonyl group); with the proviso that when A is phenylene and each R is H, then B is present; when A is -(CH)- and each R is H, then B is not -NHC(O)(CHCH)-; and when A and B are absent and each R is H, then R is not methyl. The unnatural amino acids may be in the form of a salt or may be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, and may optionally be further modified post-translationally. In some embodiments, the compound of Formula (XXXVII) is stable in aqueous solution under weakly acidic conditions for at least one month. In some embodiments, the compound of Formula (XXXVII) is stable under weakly acidic conditions for at least two weeks. In some embodiments, the compound of Formula (XXXVII) is stable under weakly acidic conditions for at least five days. In some embodiments, the acidic conditions are pH 2-8.

[0365] In certain embodiments of a compound of Formula (XXXVII), B is lower alkylene, substituted lower alkylene, —O-(alkylene or substituted alkylene)-, —C(R′)═NN(R′)-, —N(R′)CO-, —C(O)-, —C(R′)═N-, —C(O)-(alkylene or substituted alkylene)-, —CON(R′)-(alkylene or substituted alkylene)-, —S(alkylene or substituted alkylene)-, —S(O)(alkylene or substituted alkylene)-, or —S(O)(alkylene or substituted alkylene)-. In some embodiments of a compound of Formula (XXXVII), B is -O(CH2)-, -CH=N-, -CH=N-NH-, -NHCH2-, -NHCO-, -C(O)-, -C(O)-(CH2)-, -CONH-(CH2)-, -SCH2-, -S(=O)CH2-, or -S(O)2CH2-. In some embodiments of a compound of Formula (XXXVII), R is alkyl or cycloalkyl having 1 to 6 carbon atoms. In some embodiments of a compound of Formula (XXXVII), R is -CH3, -CH(CH3)2, or cyclopropyl. In some embodiments of a compound of Formula (XXXVII), R1 is H, tert-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), N-acetyl, tetrafluoroacetyl (TFA), or benzyloxycarbonyl (Cbz). In some embodiments of a compound of Formula (XXXVII), R1 is a resin, an amino acid, a polypeptide, an antibody, or a polynucleotide. In some embodiments of a compound of Formula (XXXVII), R2 is OH, O-methyl, O-ethyl, or Ot-butyl. In some embodiments of a compound of Formula (XXXVII), R2 is a resin, an amino acid, a polypeptide, an antibody, or a polynucleotide. In some embodiments of a compound of Formula (XXXVII), R2 is a polynucleotide. In some embodiments of a compound of Formula (XXXVII), R2 is ribonucleic acid (RNA).

[0366] In certain embodiments of the compound of Formula (XXXVII),

[0367] [ka]

[0368] is selected from the group consisting of: (i) A is a substituted lower alkylene, a 4-carbon arylene, a substituted arylene, a heteroarylene, a substituted heteroarylene, an alkarylene, a substituted alkarylene, an aralkylene, or a substituted aralkylene; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, —O—, —O-(alkylene or substituted alkylene)-, —S—, —S(O)—, —S(O)2-, —NS(O)2-, —OS(O)2-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —N(R′)—, —C(O)N(R′)-, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—, —N(R′)CO-(a a bivalent linker selected from the group consisting of alkylene, or substituted alkylene)-, -N(R')C(O)O-, -N(R')C(S)-, -S(O)N(R'), -S(O)N(R'), -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O)N(R')-, -N(R')S(O)N(R')-, -N(R')-N=, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')-N(R')-; (ii) A is optionally substituted lower alkylene, 4-carbon arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, if present; B is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S(O)-, -S(O)2-, -NS(O)2-, -OS(O)2-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -N(R')-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -N(R')CO-(alkylene, or is a bivalent linker selected from the group consisting of substituted alkylene)-, —N(R′)C(O)O—, —N(R′)C(S)—, —S(O)N(R′), —S(O)N(R′), —N(R′)C(O)N(R′)-, —N(R′)C(S)N(R′)-, —N(R′)S(O)N(R′)-, —N(R′)S(O)N(R′)-, —N(R′)-N=, —C(R′)=NN(R′)-, —C(R′)=NN=, —C(R′)-N=N—, and —C(R′)-N(R′)-N(R′)-N(R′)-; (iii) A is lower alkylene; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, —O—, —O-(alkylene or substituted alkylene)-, —S—, —S(O)—, —S(O)2-, —NS(O)2-, —OS(O)2-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —N(R′)—, —C(O)N(R′)-, —CSN(R′)-, —CON(R′)-(alkylene or substituted alkylene)- a bivalent linker selected from the group consisting of -N(R')C(O)O-, -N(R')C(S)-, -S(O)N(R'), -S(O)N(R'), -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O)N(R')-, -N(R')S(O)N(R')-, -N(R')-N=, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')-; (iv) A is phenylene; B is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S(O)-, -S(O)2-, -NS(O)2-, -OS(O)2-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -N(R')-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -N(R')CO-(alkylene, or is a bivalent linker selected from the group consisting of substituted alkylene)-, —N(R′)C(O)O—, —N(R′)C(S)—, —S(O)N(R′), —S(O)N(R′), —N(R′)C(O)N(R′)-, —N(R′)C(S)N(R′)-, —N(R′)S(O)N(R′)-, —N(R′)S(O)N(R′)-, —N(R′)-N=, —C(R′)=NN(R′)-, —C(R′)=NN=, —C(R′)-N=N—, and —C(R′)-N(R′)-N(R′)-N(R′)-; K is

[0369] [ka]

[0370] and each R' is independently H, alkyl, or substituted alkyl; R1 is optional and, if present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is optional and, if present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; each R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0371] Also included are amino acids having the structure of formula (XXXVIII):

[0372] [ka]

[0373] (In the formula, A is optionally, if present, lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (where k is 1, 2, or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) ka linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, at least one amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, at least one amino acid, a polypeptide, or a polynucleotide; with the proviso that when A is phenylene, B is present, and when A is -(CH)-, B is not -NHC(O)(CHCH)-, and when A and B are absent, R is not methyl. The unnatural amino acids may be in the form of a salt or may be incorporated into an unnatural amino acid polypeptide, polymer, polysaccharide, or polynucleotide, and may optionally be post-translationally modified. Also included are amino acids having the structure of formula (XXXIX):

[0374] [ka]

[0375] (In the formula, B is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (where k is 1, 2, or 3), -S(O) k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; Each R a is H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (where k is 1, 2, or 3), -C(O)N(R')2, -OR', and -S(O) k R', where each R' is independently H, alkyl, or substituted alkyl. The unnatural amino acids may be in the form of a salt or may be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, and may optionally be post-translationally modified. The following amino acids are also included:

[0376] [ka]

[0377] The unnatural amino acids may optionally be in the form of protected groups, carboxyl-protecting groups, and / or salts, or may be incorporated into polypeptides, polymers, polysaccharides, or polynucleotides of the unnatural amino acids, and may optionally be post-translationally modified.

[0378] Also included are the following amino acids having the structure of formula (XXXX):

[0379] [ka]

[0380] (In the formula, B is -NS(O)2-, -OS(O)2-, optionally, if present, lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (where k is 1, 2, or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) kN(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; Each R a is H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (where k is 1, 2, or 3), -C(O)N(R')2, -OR', and -S(O) k R', ... With the proviso that when A is -(CH)-, then B is not -NHC(O)(CHCH)-. The unnatural amino acids may be in the form of a salt or may be incorporated into an unnatural amino acid polypeptide, polymer, polysaccharide, or polynucleotide, and may optionally be post-translationally modified. The following amino acids are also included:

[0381] [ka]

[0382] wherein the compound is optionally an amino-protecting group, optionally a carboxyl-protecting group, optionally an amino-protecting group and a carboxyl-protecting group, or a salt thereof, or may be incorporated into a polypeptide, polymer, polysaccharide, or polynucleotide of a non-natural amino acid, and optionally further modified post-translationally. Also included are the following amino acids having the structure of formula (XXXXI):

[0383] [ka]

[0384] (In the formula, A is optionally, if present, lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (where k is 1, 2, or 3), -S(O) k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; R1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; The unnatural amino acids may be in the form of a salt or may be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, and may optionally be post-translationally modified. Also included are the following amino acids having the structure of formula (XXXXII):

[0385] [ka]

[0386] (In the formula, B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (where k is 1, 2, or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; Each R a is H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (where k is 1, 2, or 3), -C(O)N(R')2, -OR', and -S(O) kR' are independently selected from the group consisting of: R', where each R' is independently H, alkyl, or substituted alkyl.

[0387] The unnatural amino acids may be in the form of a salt or may be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, and may optionally be post-translationally modified. The following amino acids are also included:

[0388] [ka]

[0389] wherein the compound is optionally an amino-protecting group, optionally a carboxyl-protecting group, optionally an amino-protecting group and a carboxyl-protecting group, or a salt thereof, or may be incorporated into a polypeptide, polymer, polysaccharide, or polynucleotide of a non-natural amino acid, and optionally further modified post-translationally. Also included are the following amino acids having the structure of formula (XXXXIV):

[0390] [ka]

[0391] (In the formula, B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (where k is 1, 2, or 3), -S(O) k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; Each R a is H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (where k is 1, 2, or 3), -C(O)N(R')2, -OR', and -S(O) k R', where each R' is independently H, alkyl, or substituted alkyl; and n is 0-8.

[0392] The unnatural amino acids may be in the form of a salt or may be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, and may optionally be post-translationally modified. The following amino acids are also included:

[0393] [ka]

[0394] wherein the compound is optionally an amino-protecting group, optionally a carboxyl-protecting group, optionally an amino-protecting group and a carboxyl-protecting group, or a salt thereof, or may be incorporated into a polypeptide, polymer, polysaccharide, or polynucleotide of a non-natural amino acid, and optionally further modified post-translationally. The unnatural amino acids described herein can contain groups such as dicarbonyl groups, dicarbonyl-like groups, masked dicarbonyl groups, and protected dicarbonyl groups in addition to monocarbonyl structures.

[0395] For example, the following amino acids having the structure of formula (XXXXV) are included:

[0396] [ka]

[0397] (In the formula, A is optionally, if present, lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k - (where k is 1, 2, or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; The unnatural amino acids may be in the form of a salt or may be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, and may optionally be post-translationally modified. Also included are the following amino acids having the structure of formula (XXXXVI):

[0398] [ka]

[0399] (In the formula, B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (where k is 1, 2, or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; Each R a is H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (where k is 1, 2, or 3), -C(O)N(R')2, -OR', and -S(O)k R' are independently selected from the group consisting of: R', where each R' is independently H, alkyl, or substituted alkyl.

[0400] The unnatural amino acids may be in the form of a salt or may be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, and may optionally be post-translationally modified. The following amino acids are also included:

[0401] [ka]

[0402] wherein the compound optionally has an amino-protecting group and a carboxyl-protecting group, or a salt thereof. The unnatural amino acid may be in the form of a salt or may be incorporated into a polypeptide, polymer, polysaccharide, or polynucleotide of the unnatural amino acid, and may optionally be post-translationally modified. Also included are the following amino acids having the structure of formula (XXXXVII):

[0403] [ka]

[0404] (In the formula, B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (where k is 1, 2, or 3), -S(O) k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; Each R a is H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (where k is 1, 2, or 3), -C(O)N(R')2, -OR', and -S(O) k R', where each R' is independently H, alkyl, or substituted alkyl; and n is 0-8.

[0405] The unnatural amino acids may be in the form of a salt or may be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, and may optionally be post-translationally modified. The following amino acids are also included:

[0406] [ka]

[0407] wherein the compound optionally has amino and carboxyl protecting groups, or a salt thereof, or may be incorporated into a polypeptide, polymer, polysaccharide, or polynucleotide of the unnatural amino acid, and may optionally be post-translationally modified. Also included are the following amino acids having the structure of formula (XXXXVIII):

[0408] [ka]

[0409] (In the formula, A is optionally, if present, lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; X1 is C, S, or S(O); L is alkylene, substituted alkylene, N(R')(alkylene), or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; The unnatural amino acids may be in the form of a salt or may be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, and may optionally be post-translationally modified. Also included are the following amino acids having the structure of formula (XXXXIX):

[0410] [ka]

[0411] (In the formula, A is optionally, if present, lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; L is alkylene, substituted alkylene, N(R')(alkylene), or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; The unnatural amino acids may be in the form of a salt or may be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, and may optionally be post-translationally modified. Also included are the following amino acids having the structure of formula (XXXXX):

[0412] [ka]

[0413] (In the formula, A is optionally, if present, lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; L is alkylene, substituted alkylene, N(R')(alkylene), or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0414] The unnatural amino acids may be in the form of a salt or may be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, and may optionally be post-translationally modified. Also included are the following amino acids having the structure of formula (XXXXXI):

[0415] [ka]

[0416] (In the formula, A is optionally, if present, lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; X1 is C, S, or S(O), and n is 0, 1, 2, 3, 4, or 5; Each CR 8 R 9 Each R in the group 8 and R 9 are independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, and aryl, or any R 8 and R 9 can together form =O or cycloalkyl, or R 8 Any groups adjacent to the group can be taken together to form a cycloalkyl.

[0417] The unnatural amino acids may be in the form of a salt or may be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, and may optionally be post-translationally modified. Also included are the following amino acids having the structure of formula (XXXXXII):

[0418] [ka]

[0419] (In the formula, A is optionally, if present, lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; n is 0, 1, 2, 3, 4, or 5, and each CR 8 R 9 Each R in the group 8 and R 9 are independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, and aryl, or any R 8 and R 9 can together form =O or cycloalkyl, or R 8 Any groups adjacent to the group can be taken together to form a cycloalkyl.

[0420] The unnatural amino acids may be in the form of a salt or may be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, and may optionally be post-translationally modified. Also included are the following amino acids having the structure of formula (XXXXXIII):

[0421] [ka]

[0422] (In the formula, A is optionally, if present, lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; n is 0, 1, 2, 3, 4, or 5, and each CR 8 R 9 Each R in the group 8 and R 9 are independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, and aryl, or any R 8 and R 9 can together form =O or cycloalkyl, or R 8 Any groups adjacent to the group can be taken together to form a cycloalkyl.

[0423] The unnatural amino acids may be in the form of a salt or may be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, and may optionally be post-translationally modified. Also included are the following amino acids having the structure of formula (XXXXXIV):

[0424] [ka]

[0425] (In the formula, A is optionally, if present, lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; X1 is C, S, or S(O) and L is alkylene, substituted alkylene, N(R')(alkylene), or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0426] The unnatural amino acids may be in the form of a salt or may be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, and may optionally be post-translationally modified. Also included are the following amino acids having the structure of formula (XXXXXV):

[0427] [ka]

[0428] (In the formula, A is optionally, if present, lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; L is alkylene, substituted alkylene, N(R')(alkylene), or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0429] The unnatural amino acids may be in the form of a salt or may be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, and may optionally be post-translationally modified. Also included are the following amino acids having the structure of formula (XXXXXVI):

[0430] [ka]

[0431] (In the formula, A is optionally, if present, lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; L is alkylene, substituted alkylene, N(R')(alkylene), or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0432] The unnatural amino acids may be in the form of a salt or may be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, and may optionally be post-translationally modified. Also included are amino acids having the structure of formula (XXXXXVII).

[0433] [ka]

[0434] (In the formula, A is optionally, if present, lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; M is

[0435] [ka]

[0436] and (a) indicates a bond to the A group while (b) indicates a bond to the respective carbonyl group; or R3 and R4 are independently selected from H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; or R3 and R4, two R3 groups, or two R4 groups optionally form a cycloalkyl or heterocycloalkyl; R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; T3 is a bond, C(R)(R), O, or S, where R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; The unnatural amino acids may be in the form of a salt or may be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, and may optionally be post-translationally modified. Also included are amino acids having the structure of formula (XXXXXVIII).

[0437] [ka]

[0438] (In the formula, M is

[0439] [ka]

[0440] and (a) indicates a bond to the A group while (b) indicates a bond to the respective carbonyl group; or R3 and R4 are independently selected from H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; or R3 and R4, two R3 groups, or two R4 groups optionally form a cycloalkyl or heterocycloalkyl; R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; T3 is a bond, C(R)(R), O, or S, where R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; Each R a is H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (where k is 1, 2, or 3), -C(O)N(R')2, -OR', and -S(O) k R' are independently selected from the group consisting of: R', where each R' is independently H, alkyl, or substituted alkyl.

[0441] The unnatural amino acids may be in the form of a salt or may be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, and may optionally be post-translationally modified. Also included are amino acids having the structure of formula (XXXXXIX).

[0442] [ka]

[0443] (In the formula, R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; T3 is O or S.

[0444] The unnatural amino acids may be in the form of a salt or may be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, and may optionally be post-translationally modified. Also included are amino acids having the structure of formula (XXXXXX).

[0445] [ka]

[0446] (In the formula, R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. Also included are the following amino acids having the structure of formula (XXXXXX):

[0447] [ka]

[0448] The unnatural amino acids may be in the form of a salt or may be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, and may optionally be post-translationally modified.

[0449] Carbonyl or dicarbonyl functional groups can be selectively reacted with hydroxylamine-containing reagents in aqueous solutions under mild conditions to form the corresponding oxime bonds, which are stable in physiological environments. See, for example, Jencks, W. P., J. Am. Chem. Soc. 81, 475-481 (1959); Shao, J. and Tam, JP, J. Am. Chem. Soc. 117(14):3893-3899 (1995). Furthermore, the unique reactivity of carbonyl or dicarbonyl groups allows for selective modification in the presence of other amino acid side chains. See, e.g., Cornish, VW, et al., J. Am. Chem. Soc. 118:8150-8151 (1996); Geoghegan, KF & Stroh, JG, Bioconjug. Chem. 3:138-146 (1992); Mahal, LK, et al. See, e.g., et al., Science 276:1125-1128 (1997).

[0450] The synthesis of p-acetyl-(+ / -)-phenylalanine and m-acetyl-(+ / -)-phenylalanine is described in Zhang, Z. et al., Biochemistry 42: 6735-6746 (2003), which is incorporated by reference. Other carbonyl- or dicarbonyl-containing amino acids can be prepared similarly.

[0451] In some embodiments, polypeptides containing unnatural amino acids are chemically modified to generate reactive carbonyl or dicarbonyl functional groups. For example, aldehyde functional groups useful for conjugation reactions can be generated from functional groups bearing adjacent amino and hydroxyl groups. When the biologically active molecule is a polypeptide, for example, an N-terminal serine or threonine (which may be naturally present or exposed through chemical or enzymatic digestion) can be used to generate aldehyde functional groups under mild oxidative cleavage conditions using periodate. See, e.g., Gaertner, et al., Bioconjug. Chem. 3: 262-268 (1992); Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3: 138-146 (1992); Gaertner et al., J. Biol. Chem. 269: 7224-7230 (1994). However, methods known in the art are restricted to the amino acid at the N-terminus of a peptide or protein.

[0452] Additionally, by way of example, unnatural amino acids bearing adjacent hydroxyl and amino groups can be incorporated into polypeptides as "masked" aldehyde functional groups. For example, 5-hydroxylysine possesses an amine at the epsilon position and an adjacent hydroxyl group. Reaction conditions for generating the aldehyde include the addition of a molar excess of sodium metaperiodate in a mild environment to avoid oxidation at other sites in the polypeptide. The pH of the oxidation reaction is typically about 7.0. A typical reaction involves adding about a 1.5 molar excess of sodium metaperiodate to a buffered solution of the polypeptide, followed by incubation in the dark for about 10 minutes. See, e.g., U.S. Patent No. 6,423,685.

[0453] B. Structure and Synthesis of Unnatural Amino Acids: Dicarbonyl, Dicarbonyl-Like, Masked, and Protected Dicarbonyl Groups Amino acids having electrophilic reactive groups are capable of various reactions, particularly binding molecules via nucleophilic addition reactions. Examples of such electrophilic reactive groups include dicarbonyl groups (including diketone groups, ketoaldehyde groups, ketoacid groups, ketoester groups, and ketothioester groups), carbonyl-like or dicarbonyl-like groups (which have similar reactivity to and are structurally similar to dicarbonyl groups), masked dicarbonyl groups (which can be readily converted to dicarbonyl groups), or protected dicarbonyl groups (which, when deprotected, have similar reactivity to dicarbonyl groups). Examples of such amino acids include amino acids having the structure of formula (XXXVII):

[0454] [ka]

[0455] (In the formula, A is optionally, if present, lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional and, if present, is a linker attached at one end to the diamine-containing moiety, said linker being lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -C(O)R"-, -S(O) k-(alkylene or substituted alkylene)- (where k is 1, 2, or 3), -C(O)-(alkylene or substituted alkylene)-, -C(S)-(alkylene or substituted alkylene)-, -NR"-(alkylene or substituted alkylene)-, -CON(R")-(alkylene or substituted alkylene)-, CSN(R")-(alkylene or substituted alkylene)-, and -N(R")-CO-(alkylene or substituted alkylene)-, wherein each R" is independently H, alkyl, or substituted alkyl; K is

[0456] [ka]

[0457] and where: T1 is a bond, an optionally substituted alkylene having 1 to 4 carbon atoms, an optionally substituted alkenylene having 1 to 4 carbon atoms, or an optionally substituted heteroalkyl; each optional substituent is independently selected from lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; T2 is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (where k is 1, 2, or 3), -S(O) k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; T3 is

[0458] [ka]

[0459] and where: each X1 is independently selected from the group consisting of -O-, -S-, -N(H)-, -N(R)-, -N(Ac)-, and -N(OMe)-; and X2 is -OR, -OAc, -SR, -N(R)2, -N(R)(Ac), -N(R)(OMe), or N3, wherein each R' is independently H, alkyl, or substituted alkyl; R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, or -ABKR groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl containing at least one carbonyl group (including a dicarbonyl group), protected carbonyl group (including a protected dicarbonyl group), or masked carbonyl group (including a masked dicarbonyl group); or The -KR groups together form a monocyclic or bicyclic cycloalkyl or heterocycloalkyl containing at least one carbonyl group (including a dicarbonyl group), protected carbonyl group (including a protected dicarbonyl group), or masked carbonyl group (including a masked dicarbonyl group). Non-limiting examples of dicarbonyl amino acids having the structure of formula (XXXVII) include:

[0460] [ka] JPEG0007638942000124.jpg63169

[0461] Further included are the following amino acids having the structure of formula (XXXVII):

[0462] [ka]

[0463] The unnatural amino acids may be in the form of a salt or may be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, and may optionally be post-translationally modified.

[0464] C. Structure and Synthesis of Unnatural Amino Acids: Ketoalkyne Groups, Ketoalkyne-Like Groups, Masked Ketoalkyne Groups, Protected Ketoalkyne Groups (Groupk), Alkyne Groups, and Cycloalkyne Groups Amino acids containing reactive groups with dicarbonyl-like reactivity can link molecules via nucleophilic addition reactions. Such electrophilic reactive groups include ketoalkyne groups, ketoalkyne-like groups (which have similar reactivity to and are structurally similar to ketoalkyne groups), masked ketoalkyne groups (which can be readily converted to ketoalkyne groups), or protected ketoalkyne groups (which, when deprotected, have similar reactivity to ketoalkyne groups). In some embodiments, amino acids containing reactive groups with terminal alkynes, internal alkynes, or cycloalkynes can link molecules via cycloaddition reactions (e.g., 1,3-dipolar cycloaddition, azide-alkyne Huisgen cycloaddition, etc.). Such amino acids include amino acids having the structure of formula (XXXXXXI-A) or (XXXXXXI-B).

[0465] [ka]

[0466] (In the formula, A is optionally, if present, lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional and, if present, is a linker attached at one end to the diamine-containing moiety, the linker being selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -C(O)R"-, -S(O) k-(alkylene or substituted alkylene)- (where k is 1, 2, or 3), -C(O)-(alkylene or substituted alkylene)-, -C(S)-(alkylene or substituted alkylene)-, -NR"-(alkylene or substituted alkylene)-, -CON(R")-(alkylene or substituted alkylene)-, -CSN(R")-(alkylene or substituted alkylene)-, and -N(R")CO-(alkylene or substituted alkylene)-, wherein each R" is independently H, alkyl, or substituted alkyl; G is optional, and if present,

[0467] [ka]

[0468] and T4 is a carbonyl protecting group, and examples of the carbonyl protecting group include, but are not limited to,

[0469] [ka]

[0470] wherein each X1 is independently selected from the group consisting of -O-, -S-, -N(H)-, -N(R)-, -N(Ac)-, and -N(OMe)-; X2 is -OR, -OAc, -SR, -N(R)2, -N(R)(Ac), -N(R)(OMe), or N3; and each R' is independently H, alkyl, or substituted alkyl; R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4, or two R3 groups, may optionally form a cycloalkyl or heterocycloalkyl; R 19 are independently selected from the group consisting of alkyl having 1 to 6 carbon atoms, alkoxy having 1 to 6 carbon atoms, ester, ether, thioether, aminoalkyl, halogen, alkyl ester, aryl ester, amide, aryl amide, alkyl halide, alkylamine, alkylsulfonic acid, alkylnitro, thioester, sulfonyl ester, halosulfonyl, nitrile, alkylnitrile, and nitro; q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.) D. Structure and Synthesis of Unnatural Amino Acids: Ketoamine, Ketoamine-Like, Masked Ketoamine, and Protected Ketoamine Groups Amino acids containing reactive groups with dicarbonyl-like reactivity can bind molecules via nucleophilic addition reactions. Such reactive groups include ketoamine groups, ketoamine-like groups (which have similar reactivity to and are structurally similar to ketoamine groups), masked ketoamine groups (which can be readily converted to ketoamine groups), or protected ketoamine groups (which, when deprotected, have similar reactivity to ketoamine groups). Such amino acids include amino acids having the structure of formula (XXXXXXII).

[0471] [ka]

[0472] (In the formula, A is optionally, if present, lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional and, if present, is a linker attached at one end to the diamine-containing moiety, the linker being selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -C(O)R"-, -S(O) k -(alkylene or substituted alkylene)- (where k is 1, 2, or 3), -C(O)-(alkylene or substituted alkylene)-, -C(S)-(alkylene or substituted alkylene)-, -NR"-(alkylene or substituted alkylene)-, -CON(R")-(alkylene or substituted alkylene)-, -CSN(R")-(alkylene or substituted alkylene)-, and -N(R")CO-(alkylene or substituted alkylene)-, wherein each R" is independently H, alkyl, or substituted alkyl; G is

[0473] [ka]

[0474] and T1 is an optionally substituted alkylene having 1 to 4 carbon atoms, an optionally substituted alkenylene having 1 to 4 carbon atoms, or an optionally substituted heteroalkyl; T4 is a carbonyl protecting group, and examples of the carbonyl protecting group include, but are not limited to,

[0475] [ka]

[0476] wherein each X1 is independently selected from the group consisting of -O-, -S-, -N(H)-, -N(R')-, -N(Ac)-, and -N(OMe)-; X2 is -OR, -OAc, -SR', -N(R')2, -N(R')(Ac), -N(R')(OMe), or N3; and each R' is independently H, alkyl, or substituted alkyl; R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; Each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4, or two R3 groups, may optionally form a cycloalkyl or heterocycloalkyl. Amino acids having the structure of chemical formula (XXXXXXII) include amino acids having the structures of chemical formula (XXXXXXIII) and chemical formula (XXXXXXIV).

[0477] [ka]

[0478] (In the formula, each R a are independently H, halogen, alkyl, substituted alkyl, -N(R')2, -C(O) k R' (where k is 1, 2, or 3), -C(O)N(R')2, -OR', and -S(O) k R', where each R' is independently H, alkyl, or substituted alkyl. E. Structure and Synthesis of Unnatural Amino Acids: Diamines, Diamine-like, Masked Diamines, Protected Amines, and Azides Amino acids with nucleophilic reactive groups allow for various reactions to link molecules with other molecules via electrophilic addition reactions. Examples of such nucleophilic reactive groups include diamine groups (including hydrazine groups, amidine groups, imine groups, 1,1-diamine groups, 1,2-diamine groups, 1,3-diamine groups, and 1,4-diamine groups), diamine-like groups (which have similar reactivity to and are structurally similar to diamine groups), masked diamine groups (which can be readily converted to diamine groups), or protected diamine groups (which have similar reactivity to diamine groups when deprotected). In some embodiments, amino acids containing azide-containing reactive groups can link molecules via cycloaddition reactions (e.g., 1,3-dipolar cycloaddition, azide-alkyne Huisgen cycloaddition, etc.).

[0479] In another aspect, methods for chemical synthesis of hydrazine-substituted molecules for derivatization of carbonyl-substituted dolastatin derivatives are described. In certain embodiments, the hydrazine-substituted molecules can be dolastatin-linked derivatives. In certain embodiments, methods for producing hydrazine-substituted molecules are described. The methods are suitable, for example, for derivatizing carbonyl-containing non-natural amino acid polypeptides, including, by way of example only, ketone-containing non-natural amino acid polypeptides or aldehyde-containing non-natural amino acid polypeptides. In further or additional embodiments, the non-natural amino acid is site-specifically incorporated during in vivo protein translation. In further or additional embodiments, the hydrazine-substituted dolastatin derivatives can site-specifically derivatize carbonyl-containing non-natural amino acids via nucleophilic attack of each carbonyl group to form heterocyclic-derivatized polypeptides, such as nitrogen-containing heterocyclic-derivatized polypeptides, in a site-specific manner. In further or additional embodiments, methods for making hydrazine-substituted dolastatin derivatives provide access to a wide variety of site-specifically derivatized polypeptides. In further or additional embodiments, methods for synthesizing hydrazine-functionalized polyethylene glycol (PEG)-linked dolastatin derivatives are described.

[0480] Such amino acids include those having the structure of chemical formula (XXXVII-A) or (XXXVII-B).

[0481] [ka]

[0482] (In the formula, A is optionally, if present, lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional and, if present, is a linker attached at one end to the diamine-containing moiety, said linker being selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -C(O)R"-, -C(O)R"-, -S(O) k -(alkylene or substituted alkylene)- (where k is 1, 2, or 3), -C(O)-(alkylene or substituted alkylene)-, -C(S)-(alkylene or substituted alkylene)-, -NR"-(alkylene or substituted alkylene)-, -CON(R")-(alkylene or substituted alkylene)-, -CSN(R")-(alkylene or substituted alkylene)-, and -N(R")CO-(alkylene or substituted alkylene)-, wherein each R" is independently H, alkyl, or substituted alkyl; K is

[0483] [ka]

[0484] (In the formula, R8 and R9 are independently selected from H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, or an amine protecting group; T1 is a bond, an optionally substituted alkylene having 1 to 4 carbon atoms, an optionally substituted alkenylene having 1 to 4 carbon atoms, or an optionally substituted heteroalkyl; T2 is an optionally substituted alkylene having 1 to 4 carbon atoms, an optionally substituted alkenylene having 1 to 4 carbon atoms, an optionally substituted heteroalkyl, an optionally substituted aryl, or an optionally substituted heteroaryl; wherein each optional substituent is independently selected from lower alkyl, substituted lower alkyl, lower cycloalkyl, substituted lower cycloalkyl, lower alkenyl, substituted lower alkenyl, alkynyl, lower heteroalkyl, substituted heteroalkyl, lower heterocycloalkyl, substituted lower heterocycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; Each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4, or two R3 groups, may optionally form a cycloalkyl or heterocycloalkyl, or -ABKR groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl having at least one diamine group, protected diamine group, or masked diamine group, where at least one amine group on -ABKR is an optionally protected amine, or the -BKR groups together form a bicyclic or tricyclic cycloalkyl or cycloaryl or heterocycloalkyl having at least one diamine group, protected diamine group or masked diamine group; or The -KR groups together form a monocyclic or bicyclic cycloalkyl or heterocycloalkyl having at least one diamine group, protected diamine group, or masked diamine group. is.

[0485] In some embodiments, there is a compound having the following Structure 1 or Structure 2:

[0486] [ka]

[0487] (In the formula, A is optionally, if present, lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional and, if present, is a linker attached at one end to the diamine-containing moiety, the linker being selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -C(O)R"-, -S(O) k -(alkylene or substituted alkylene)- (where k is 1, 2, or 3), -C(O)-(alkylene or substituted alkylene)-, -C(S)-(alkylene or substituted alkylene)-, -NR"-(alkylene or substituted alkylene)-, -CON(R")-(alkylene or substituted alkylene)-, -CSN(R")-(alkylene or substituted alkylene)-, and -N(R")CO-(alkylene or substituted alkylene)-, wherein each R" is independently H, alkyl, or substituted alkyl; T1 is a bond or CH2 and T2 is CH; wherein each optional substituent is independently selected from lower alkyl, substituted lower alkyl, lower cycloalkyl, substituted lower cycloalkyl, lower alkenyl, substituted lower alkenyl, alkynyl, lower heteroalkyl, substituted heteroalkyl, lower heterocycloalkyl, substituted lower heterocycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl; R1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; Each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4, or two R3 groups, may optionally form a cycloalkyl or heterocycloalkyl, or the -AB-diamine-containing moieties together form a bicyclic cycloalkyl or heterocycloalkyl having at least one diamine group, a protected diamine group, or a masked diamine group, wherein at least one amine group of the -AB-diamine-containing moiety is an optionally protected amine or is an active metabolite, salt, or pharmaceutically acceptable prodrug or solvate thereof; or -B- The groups consisting of the diamine-containing moiety together form a bicyclic or tricyclic cycloalkyl or cycloaryl or heterocycloalkyl having at least one diamine group, protected diamine group, or masked diamine group. Non-limiting examples of amino acids having the structure of formula (XXXVII) include:

[0488] [ka]

[0489] The unnatural amino acids may be in the form of a salt, or may be incorporated into a unnatural amino acid polypeptide, polymer, polysaccharide, or polynucleotide, and / or may optionally be post-translationally modified.

[0490] In some embodiments, the compound of Formula (XXXVII) is stable in aqueous solution under weakly acidic conditions for at least one month. In some embodiments, the compound of Formula (XXXVII) is stable under weakly acidic conditions for at least two weeks. In some embodiments, the compound of Formula (XXXVII) is stable under weakly acidic conditions for at least five days. In some embodiments, the acidic conditions are a pH of about 2 to 8.

[0491] In certain embodiments of a compound of Formula (XXXVII), B is lower alkylene, substituted lower alkylene, O-(alkylene or substituted alkylene)-, C(R')=NN(R')-, -N(R')CO-, C(O)-, -C(R')=N-, C(O)-(alkylene or substituted alkylene)-, CON(R')(alkylene or substituted alkylene)-, -S(alkylene or substituted alkylene)-, -S(O)(alkylene or substituted alkylene)-, or -S(O)(alkylene or substituted alkylene)-. In some embodiments of a compound of Formula (XXXVII), B is -O(CH2)-, -CH=N-, CH=NNH-, -NHCH2-, -NHCO-, C(O)-, C(O)(CH2)-, CONH(CH2)-, -SCH2-, -S(=O)CH2-, or -S(O)2CH2-. In some embodiments of a compound of Formula (XXXVII), R is alkyl or cycloalkyl having 1 to 6 carbon atoms. In some embodiments of a compound of Formula (XXXVII), R is -CH3, -CH(CH3)2, or cyclopropyl. In some embodiments of a compound of Formula (XXXVII), R1 is H, tert-butyloxycarbonyl (t-Boc), 9-fluorenylmethoxycarbonyl (Fmoc), N-acetyl, tetrafluoroacetyl (TFA), or benzyloxycarbonyl (Cbz). In some embodiments of a compound of Formula (XXXVII), R1 is a resin, an amino acid, a polypeptide, or a polynucleotide. In some embodiments of a compound of Formula (XXXVII), R1 is an antibody, an antibody fragment, or a monoclonal antibody. In some embodiments of a compound of Formula (XXXVII), R2 is OH, O-methyl, O-ethyl, or Ot-butyl. In some embodiments of a compound of Formula (XXXVII), R2 is a resin, at least one amino acid, a polypeptide, or a polynucleotide. In some embodiments of a compound of Formula (XXXVII), R2 is an antibody, an antibody fragment, or a monoclonal antibody.

[0492] Non-limiting examples of amino acids having the structure of formula (XXXVII) also include:

[0493] [ka]

[0494] Non-limiting examples of protected amino acids having the structure of formula (XXXVII) include:

[0495] [ka]

[0496] F. Structure and Synthesis of Unnatural Amino Acids: Aromatic Amines Unnatural amino acids with nucleophilic reactive groups allow for a variety of reactions to link molecules together, including, but not limited to, reductive alkylation reactions using aldehyde-containing dolastatin linker derivatives. Unnatural amino acids with such nucleophilic reactive groups include, by way of example only, aromatic amine groups (including secondary and tertiary amine groups), masked aromatic amine groups (which can be readily converted to aromatic amine groups), or protected aromatic amine groups (which, when deprotected, have the same reactivity as aromatic amine groups). Examples of such unnatural amino acids containing aromatic amines include amino acids having the structure of formula (XXXXXXV).

[0497] [ka]

[0498] (In the formula,

[0499] [ka]

[0500] is selected from the group consisting of monocyclic aryl rings, bicyclic aryl rings, polycyclic aryl rings, monocyclic heteroaryl rings, bicyclic heteroaryl rings, and polycyclic heteroaryl rings; A is independently a , or N, B is independently a , N, O, or , S; Each R a are independently H, halogen, alkyl, -NO2, -CN, substituted alkyl, -N(R')2, -C(O) k R', -C(O)N(R')2, -OR', and -S(O) k R', where k is 1, 2, or 3; and n is 0, 1, 2, 3, 4, 5, or 6; R1 is H, an amino protecting group, a resin, at least one amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, at least one amino acid, a polypeptide, or a polynucleotide; each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4, or two R3 groups, may optionally form a cycloalkyl or heterocycloalkyl; M is H or -CH2R5, or the MNC(R5) moiety may form a 4- to 7-membered ring structure; R5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkyleneoxide, substituted polyalkyleneoxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -C(O)R", -C(O)OR", -C(O)N(R")2, -C(O)NHCH(R")2, -(alkylene or substituted alkylene)-N(R")2, -(alkenylene or substituted alkenylene)-N(R")2, -(alkylene or substituted alkylene)-N(R")2, -(alkylene or substituted alkylene)-ON(R"), -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-SS-(aryl or substituted aryl), where each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or -C(O)OR'; or Two R5 groups may optionally form a cycloalkyl or heterocycloalkyl, or R5 and any R a and optionally form a cycloalkyl or heterocycloalkyl; Each R' is independently H, alkyl, or substituted alkyl.) The unnatural amino acids described above can also be in the form of a salt or incorporated into an unnatural amino acid polypeptide, polymer, polysaccharide, or polynucleotide, and optionally reductively alkylated. The following structure (shown in all examples herein):

[0501] [ka]

[0502] are "A", "B", "NH-M" and "R a Although the relative orientation of the four components of the structure is not shown, these four components of the structure can be oriented (along with other components of the structure) in any chemically suitable manner, as shown in the examples herein.

[0503] Unnatural amino acids containing an aromatic amine moiety having the structure of formula (A) include unnatural amino acids having the structure:

[0504] [ka]

[0505] wherein each A' is independently CR a , N, or

[0506] [ka]

[0507] and up to two A' are selected from

[0508] [ka]

[0509] and the remaining A' is CR a , or N.) The unnatural amino acids may also be in the form of a salt or may be incorporated into an unnatural amino acid polypeptide, polymer, polysaccharide, or polynucleotide, and optionally reductively alkylated.

[0510] Non-limiting examples of unnatural amino acids containing an aromatic amine moiety having the structure of formula (XXXXXXV) include unnatural amino acids having the structure of formula (XXXXXXVI) and formula (XXXXXXVII).

[0511] [ka]

[0512] (wherein G is an amine protecting group, including, but not limited to, the following:)

[0513] [ka]

[0514] The unnatural amino acids described above can be in the form of a salt or can be incorporated into an unnatural amino acid polypeptide, polymer, polysaccharide, or polynucleotide, and optionally reductively alkylated.

[0515] The unnatural amino acid containing an aromatic amine moiety has the following structure:

[0516] [ka]

[0517] (In the formula, each R a are independently H, halogen, alkyl, -NO2, -CN, substituted alkyl, -N(R')2, -C(O) k R', -C(O)N(R')2, -OR', and -S(O) k R' (wherein k is 1, 2, or 3); M is H or -CH2R5, or the MNC(R5) moiety may form a 4- to 7-membered ring structure; R1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; R5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkyleneoxide, substituted polyalkyleneoxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -C(O)R", -C(O)OR", -C(O)N(R")2, -C(O)NHCH(R")2, -(alkylene or substituted alkylene)-N(R")2, -(alkenylene or substituted alkenylene)-N(R")2, -(alkylene or substituted alkylene)-N(R")2, -(alkylene or substituted alkylene)-ON(R"), -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-SS-(aryl or substituted aryl), where each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or -C(O)OR'; or R5 and any R a and optionally form a cycloalkyl or heterocycloalkyl; Each R' is independently H, alkyl, or substituted alkyl. The unnatural amino acids may also be in the form of a salt or may be incorporated into an unnatural amino acid polypeptide, polymer, polysaccharide, or polynucleotide.

[0518] The unnatural amino acids of formula (XXXXXXV) can be formed by reduction of a protected or masked amine moiety on the aromatic portion of the unnatural amino acid. Such protected or masked amine moieties include, but are not limited to, imine, hydrazine, nitro, or azide substituents. Reducing agents used to reduce the protected or masked amine moieties include, but are not limited to, TCEP, NaS, NaSO, LiAlH, NaBH, or NaBCNH.

[0519] (V. Non-natural amino acid-linked dolastatin derivatives) Other embodiments described herein are methods, strategies, and techniques for incorporating at least one of the above-described dolastatin linker derivatives into an unnatural amino acid. These embodiments include methods for producing, purifying, characterizing, and using the above-described dolastatin linker derivatives containing at least one of the above-described unnatural amino acids. Additionally, these embodiments include compositions of oligonucleotides (including DNA and RNA) that can be used to, at least in part, produce dolastatin linker derivatives containing at least one unnatural amino acid, and methods for producing, purifying, characterizing, and using the oligonucleotides. Additionally, these embodiments include compositions of cells capable of expressing the above-described oligonucleotides that can be used to, at least in part, produce dolastatin linker derivatives containing at least one unnatural amino acid, and methods for producing, purifying, characterizing, and using the cells.

[0520] Thus, provided and described herein are dolastatin linker derivatives having at least one unnatural amino acid or modified unnatural amino acid bearing a carbonyl, dicarbonyl, alkyne, cycloalkyne, azide, oxime, or hydroxylamine group. In certain embodiments, the dolastatin linker derivatives having at least one unnatural amino acid or modified unnatural amino acid bearing a carbonyl, dicarbonyl, alkyne, cycloalkyne, azide, oxime, or hydroxylamine group include at least one post-translational modification at a position on the polypeptide. In some embodiments, the co-translational or post-translational modification occurs via cellular mechanisms (e.g., glycosylation, acetylation, acylation, lipid modification, palmitoylation, palmitate addition, phosphorylation, glycolipid linkage modification, etc.). In many cases, the co- or post-translational modifications based on the cellular machinery occur at naturally occurring amino acid sites on the polypeptide, but in certain embodiments, the co- or post-translational modifications based on the cellular machinery occur at non-naturally occurring amino acid sites on the polypeptide.

[0521] In another embodiment, the post-translational modification does not utilize cellular machinery, but instead is functionalized by attaching a molecule (a polymer, a water-soluble polymer, a derivative of polyethylene glycol, a second protein or polypeptide or polypeptide mimetic, an antibody or antibody fragment, and any combination thereof) having a second reactive group to at least one unnatural amino acid bearing a first reactive group (including, but not limited to, an unnatural amino acid containing a ketone, aldehyde, acetal, hemiacetal, alkyne, cycloalkyne, azide, oxime, or hydroxylamine functional group) using chemical procedures described herein or appropriate for the particular reactive group. In certain embodiments, the co- or post-translational modification occurs in vivo in a eukaryotic or non-eukaryotic cell. In certain embodiments, the post-translational modification occurs in vivo without utilizing cellular machinery. Also included in these embodiments are methods for making, purifying, characterizing, and using the dolastatin linker derivatives described above that contain at least one of the translationally or post-translationally modified unnatural amino acids described above.

[0522] Additionally, the scope of the methods, compositions, strategies, and techniques described herein includes reagents capable of reacting with dolastatin linker derivatives (containing carbonyl or dicarbonyl groups, oxime groups, alkynes, cycloalkynes, azides, hydroxylamine groups, or masked or protected forms thereof) that are part of a polypeptide to produce any of the above post-translational modifications. In certain embodiments, the resulting post-translationally modified dolastatin linker derivatives contain at least one oxime group. The resulting modified oxime-containing dolastatin linker derivatives can then be subjected to subsequent modification reactions. Also included in these aspects are methods for producing, purifying, characterizing, and using the above reagents capable of any of the above post-translational modifications of the dolastatin linker derivatives.

[0523] In some embodiments, the polypeptide or non-natural amino acid-linked dolastatin derivative comprises at least one co- or post-translational modification made in vivo by one host cell that is not normally made by another host cell. In some embodiments, the polypeptide comprises at least one co- or post-translational modification made in vivo by a eukaryotic cell that is not normally made by a non-eukaryotic cell. Examples of co- or post-translational modifications include, but are not limited to, glycosylation, acetylation, acylation, lipo-modification, palmitoylation, palmitate addition, phosphorylation, glycolipid-linkage modification, and the like. In some embodiments, the co- or post-translational modification comprises attachment of an oligosaccharide to an aspartate via a GlcNAc-aspartate linkage (including, but not limited to, when the oligosaccharide has a structure such as (GlcNAc-Man)2-Man-GlcNAc-GlcNAc). In another embodiment, the co- or post-translational modification comprises attachment of an oligosaccharide (including, but not limited to, Gal-GalNAc, Gal-GlcNAc, etc.) to serine or threonine via a GalNAc-serine, GalNAc-threonine, GlcNAc-serine, or GlcNAc-threonine linkage. In certain embodiments, the protein or polypeptide can comprise a secretion or localization sequence, an epitope tag, a FLAG tag, a polyhistidine tag, a GST fusion, etc. Further included in this aspect are methods for producing, purifying, characterizing, and using the polypeptides containing at least one of the co- or post-translational modifications. In another embodiment, the glycosylated non-natural amino acid polypeptide is produced in an unglycosylated form.Non-glycosylated forms of the glycosylated non-natural amino acid polypeptides may be produced by a method comprising the following steps, or any combination of the following steps: chemical or enzymatic removal of oligosaccharide groups from an isolated, substantially purified, or non-purified glycosylated non-natural amino acid polypeptide; production of the non-natural amino acid polypeptide in a host that does not glycosylate the non-natural amino acid polypeptide (including prokaryotic or eukaryotic organisms that have been modified or mutated so as not to glycosylate the polypeptide); or introduction of a glycosylation inhibitor into a polypeptide cell culture medium in which the non-natural amino acid polypeptide is produced by a eukaryotic organism that normally glycosylates the polypeptide. Further described herein are non-glycosylated forms of normally glycosylated non-natural amino acid polypeptides (by normally glycosylated, it is meant a polypeptide that would be glycosylated if the naturally occurring polypeptide were produced in a glycosylated state). Of course, such non-glycosylated forms of normally glycosylated non-natural amino acid polypeptides (or indeed any polypeptides described herein) can be in unpurified, substantially purified, or isolated form.

[0524] In one embodiment, the non-natural amino acid polypeptide comprises one post-translational modification that is made in the presence of a reaction facilitator, and the post-translational modification is stoichiometric, stoichiometric-like, or near-stoichiometric. In another embodiment, the polypeptide is contacted with a reagent of formula (XIX) in the presence of a reaction facilitator. In another embodiment, the reaction facilitator is selected from the group consisting of:

[0525] [ka]

[0526] A. Chemical Synthesis of Unnatural Amino Acid-Linked Dolastatin Derivatives: Oxime-Containing Dolastatin Derivatives Unnatural amino acid dolastatin-linked derivatives containing an oxime group can be reacted with a variety of reagents containing a reactive carbonyl or dicarbonyl group (including, but not limited to, ketones, aldehydes, or other groups with similar reactivity) to form new unnatural amino acids bearing new oxime groups. Such oxime exchange reactions allow for further functionalization of the dolastatin-linked derivatives. Furthermore, the original dolastatin-linked derivatives containing an oxime group will be useful in that capacity, so long as the oxime bond is stable under the conditions required for incorporating the amino acid into a polypeptide (e.g., in vivo, in vitro, or chemical synthesis methods described herein).

[0527] Thus, in certain embodiments described herein, there are unnatural amino acid dolastatin-linked derivatives having side chains with oxime groups, oxime-like groups (which have similar reactivity to and are structurally similar to oxime groups), masked oxime groups (which can be readily converted to oxime groups), or protected oxime groups (which, when deprotected, have similar reactivity to oxime groups).

[0528] Such unnatural amino acid dolastatin-linked derivatives include dolastatin-linked derivatives having the structure of formula (VIII) or (IX).

[0529] [ka]

[0530] (In the formula, A is optionally, if present, lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (where k is 1, 2, or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, at least one amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, at least one amino acid, a polypeptide, or a polynucleotide; R3 and R4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4, or two R3 groups, may optionally form a cycloalkyl or heterocycloalkyl; Z has the following structure:

[0531] [ka]

[0532] R5 is H, COR8, alkyl having 1 to 6 carbon atoms, or thiazole; R8 is OH; R6 is OH or H; Ar is phenyl or pyridine; R7 is alkyl having 1 to 6 carbon atoms or hydrogen; L is alkylene-, -alkylene-C(O)-, -(alkylene-O) n -Alkylene-, -(alkylene-O) n -Alkylene-C(O)-, -(Alkylene-O) n -(CH2) n’ -NHC(O)-(CH2) n’’ -C(Me)2-SS-(CH2) n’’’ -NHC(O)-(alkylene-O) n’’’’ -Alkylene, -(alkylene-O) n -Alkylene-W-, -Alkylene-C(O)-W-, -(Alkylene-O) n -alkylene-U-alkylene-C(O)-, and -(alkylene-O) n -alkylene-U-alkylene; W has the following structure:

[0533] [ka]

[0534] U has the structure:

[0535] [ka]

[0536] n, n', n'', n''', and n'''' are each independently an integer greater than or equal to 1, or It is an active metabolite or an active pharmaceutically acceptable prodrug, or a solvate thereof.

[0537] In some embodiments of compounds of Formulas (VIII) and (IX), R5 is thiazole. In some embodiments of compounds of Formulas (VIII) and (IX), R6 is H. In some embodiments of compounds of Formulas (VIII) and (IX), Ar is phenyl. In some embodiments of compounds of Formulas (VIII) and (IX), R7 is methyl. In some embodiments of compounds of Formulas (VIII) and (IX), n is an integer from 0 to 20. In some embodiments of compounds of Formulas (VIII) and (IX), n is an integer from 0 to 10. In some embodiments of compounds of Formulas (VIII) and (IX), n is an integer from 0 to 5.

[0538] In some embodiments of compounds of Formula (VIII) and (IX), R5 is thiazole. In some embodiments of compounds of Formula (VIII) and (IX), R5 is hydrogen. In some embodiments of compounds of Formula (VIII) and (IX), R5 is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or hexyl.

[0539] In some embodiments of compounds of Formula (VIII) and (IX), R5 is -NH-(alkylene-O) nand the alkylene is -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, or -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-. In certain embodiments of compounds of Formulas (VIII) and (IX), the alkylene is methylene, ethylene, propylene, butylene, pentylene, hexylene, or heptylene.

[0540] In certain embodiments of compounds of Formula (VIII) and (IX), R8 is -NH-(alkylene-O) n -NH2, and n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

[0541] In some embodiments of compounds of Formula (VIII) and (IX), R6 is hydrogen.

[0542] In some embodiments of compounds of Formula (VIII) and (IX), Ar is phenyl.

[0543] In some embodiments of compounds of Formula (VIII) and (IX), R7 is methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, pentyl, or hexyl. In some embodiments of compounds of Formula (VIII) and (IX), R7 is hydrogen.

[0544] In some embodiments of compounds of Formula (VIII) and (IX), each L is independently a cleavable linker or a non-cleavable linker. In some embodiments of compounds of Formula (VIII) and (IX), each L is independently an oligo(ethylene glycol)-derivatized linker.

[0545] In certain embodiments of compounds of Formulas (VIII) and (IX), alkylene, alkylene', alkylene'', and alkylene''' are each independently -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, or -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-. In some embodiments of compounds of Formula (VIII) and (IX), alkylene is methylene, ethylene, propylene, butylene, pentylene, hexylene, or heptylene.

[0546] In certain embodiments of compounds of Formula (VIII) and (IX), n, n', n'', n''', and n''' are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, , 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

[0547] In some embodiments of a compound of Formula (VIII) or (IX), R1 is a polypeptide. In some embodiments of a compound of Formula (VIII) or (IX), R2 is a polypeptide. In some embodiments of a compound of Formula (VIII) or (IX), the polypeptide is an antibody. In some embodiments of a compound of Formula (VIII) or (IX), the antibody is Herceptin.

[0548] Such unnatural amino acid dolastatin conjugate derivatives include dolastatin conjugate derivatives having the structure of formula (X), (XI), (XII), or (XIII).

[0549] [ka] JPEG0007638942000154.jpg94169

[0550] (In the formula, A is optionally, if present, lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (where k is 1, 2, or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, at least one amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, at least one amino acid, a polypeptide, or a polynucleotide; R3 and R4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4, or two R3 groups, may optionally form a cycloalkyl or heterocycloalkyl; Z has the following structure:

[0551] [ka]

[0552] R5 is H, COR8, alkyl having 1 to 6 carbon atoms, or thiazole; R8 is OH; R6 is OH or H; Ar is phenyl or pyridine; R7 is alkyl having 1 to 6 carbon atoms or hydrogen; L1, L2, L3, and L4 each independently represent a bond, -alkylene-, or -(alkylene-O) n -Alkylene-J-, -Alkylene'-J-(Alkylene-O) n -Alkylene-, -J-(Alkylene-O) n -Alkylene-, -(Alkylene-O) n -Alkylene-J-(Alkylene-O) n '-Alkylene-J'-, -(Alkylene-O) n -Alkylene-J-alkylene'-, -W-, -Alkylene-W-, Alkylene'-J-(Alkylene-NMe) n -Alkylene-W-, -J-(Alkylene-NMe) na linker selected from the group consisting of -alkylene-W-, -J-alkylene-NMe-alkylene'-NMe-alkylene''-W-, and -alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene''''-W-; W has the following structure:

[0553] [ka]

[0554] Each J and J' independently has the structure:

[0555] [ka]

[0556] n and n are each independently an integer of 1 or greater.

[0557] In some embodiments of a compound of Formula (X), (XI), (XII), or (XIII), R5 is thiazole. In some embodiments of a compound of Formula (X), (XI), (XII), or (XIII), R5 is hydrogen. In some embodiments of a compound of Formula (X), (XI), (XII), or (XIII), Ar is phenyl. In some embodiments of a compound of Formula (X), (XI), (XII), or (XIII), R7 is methyl. In some embodiments of a compound of Formula (X), (XI), (XII), or (XIII), n and n' are integers from 0 to 20. In some embodiments of a compound of Formula (X), (XI), (XII), or (XIII), n and n' are integers from 0 to 10. In some embodiments of a compound of Formula (X), (XI), (XII), or (XIII), n and n' are integers from 0 to 5.

[0558] In some embodiments of a compound of Formula (X), (XI), (XII), or (XIII), R5 is thiazole. In some embodiments of a compound of Formula (X), (XI), (XII), or (XIII), R5 is hydrogen. In some embodiments of a compound of Formula (X), (XI), (XII), or (XIII), R5 is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or hexyl. In some embodiments of a compound of Formula (X), (XI), (XII), or (XIII), R5 is -NH-(alkylene-O) n and the alkylene is -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, or -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-. In certain embodiments of a compound of Formula (X), (XI), (XII), or (XIII), said alkylene is methylene, ethylene, propylene, butylene, pentylene, hexylene, or heptylene.

[0559] In certain embodiments of a compound of Formula (X), (XI), (XII), or (XIII), R5 is -NH-(alkylene-O) n-NH2, and n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

[0560] In certain embodiments of a compound of Formula (X), (XI), (XII), or (XIII), R6 is hydrogen. In some embodiments of a compound of Formula (I), R6 is hydroxy.

[0561] In certain embodiments of a compound of Formula (X), (XI), (XII), or (XIII), Ar is phenyl.

[0562] In some embodiments of a compound of Formula (X), (XI), (XII), or (XIII), R7 is methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, pentyl, or hexyl. In some embodiments of a compound of Formula (I), R7 is hydrogen.

[0563] In some embodiments of a compound of Formula (X), (XI), (XII), or (XIII), L1, L2, L3, and L4 are each independently a cleavable or non-cleavable linker. In some embodiments of a compound of Formula (I), L1, L2, L3, and L4 are each independently an oligo(ethylene glycol)-derivatized linker.

[0564] In certain embodiments of a compound of Formula (X), (XI), (XII), or (XIII), alkylene, alkylene', alkylene'', and alkylene''' are each independently -CH-, -CHCH-, -CHCHCH-, -CHCHCHCH-, -CHCHCHCHCH-, -CHCHCHCHCHCH-, -CHCHCHCHCHCHCH-, -CHCH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, or -CHCH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-. In some embodiments of a compound of Formula (X), (XI), (XII), or (XIII), alkylene is methylene, ethylene, propylene, butylene, pentylene, hexylene, or heptylene.

[0565] In certain embodiments of a compound of Formula (X), (XI), (XII), or (XIII), n and n′ are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 , 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

[0566] In some embodiments of a compound of Formula (X), (XI), (XII), or (XIII), R1 is a polypeptide. In some embodiments of a compound of Formula (X), (XI), (XII), or (XIII), R2 is a polypeptide. In some embodiments of a compound of Formula (X), (XI), (XII), or (XIII), the polypeptide is an antibody. In some embodiments of a compound of Formula (X), (XI), (XII), or (XIII), the antibody is Herceptin.

[0567] In some embodiments, a compound of Formula (X), (XI), (XII), or (XIII) is stable in aqueous solution under mildly acidic conditions for at least one month. In some embodiments, a compound of Formula (X), (XI), (XII), or (XIII) is stable under mildly acidic conditions for at least two weeks. In some embodiments, a compound of Formula (X), (XI), (XII), or (XIII) is stable under mildly acidic conditions for at least five days. In some embodiments, the acidic conditions are a pH of about 2 to 8. The unnatural amino acids can be in the form of a salt or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, optionally post-translationally modified.

[0568] Oxime-based unnatural amino acids may be synthesized by methods previously described in the art or described herein, including: (a) reacting a hydroxylamine-containing unnatural amino acid with a carbonyl- or dicarbonyl-containing reagent, (b) reacting a carbonyl- or dicarbonyl-containing unnatural amino acid with a hydroxylamine-containing reagent, or (c) reacting an oxime-containing unnatural amino acid with certain carbonyl- or dicarbonyl-containing reagents.

[0569] B. Chemical Structure and Synthesis of Unnatural Amino Acid-Linked Dolastatin Derivatives: Alkylated Aromatic Amine-Linked Dolastatin Derivatives One aspect is a dolastatin linker derivative for chemical derivatization of unnatural amino acids based on the reactivity of aromatic amine groups. In further or additional embodiments, at least one of the unnatural amino acids described above is incorporated into the dolastatin linker derivative. That is, such embodiments are unnatural amino acid-linked dolastatin derivatives. In further or additional embodiments, the dolastatin linker derivative is functionalized at the side chain to form an amine bond upon reaction with a derivatized unnatural amino acid. In further or additional embodiments, the dolastatin linker derivative is selected from dolastatin linker derivatives having aromatic amine side chains. In further or additional embodiments, the dolastatin linker derivative comprises a masked side chain, including a masked aromatic amine group. In further or additional embodiments, the unnatural amino acid is selected from amino acids having aromatic amine side chains. In further or additional embodiments, the unnatural amino acid comprises a masked side chain, including a masked aromatic amine group.

[0570] Other aspects are carbonyl-substituted dolastatin linker derivatives, such as aldehydes and ketones, for the production of derivatized non-natural amino acid polypeptides based on amine bonds. A further embodiment is an aldehyde-substituted dolastatin linker derivative used to derivatize an aromatic amine-containing non-natural amino acid polypeptide by forming an amine bond between the derivatized dolastatin linker and the aromatic amine-containing non-natural amino acid polypeptide.

[0571] In further or additional embodiments, the unnatural amino acid comprises an aromatic amine side chain, and the aromatic amine is selected from an aryl amine or a heteroaryl amine. In further or additional embodiments, the unnatural amino acid is similar in structure to a natural amino acid but contains an aromatic amine group. In other or further embodiments, the unnatural amino acid is similar to phenylalanine or tyrosine (aromatic amino acids). In one embodiment, the unnatural amino acid has properties that differ from those of a natural amino acid. In one embodiment, such properties are the chemical reactivity of the side chain. In further embodiments, this different chemical reactivity allows the side chain of the unnatural amino acid to undergo reactions while still a unit of a polypeptide, unlike the side chains of naturally occurring amino acid units in the same polypeptide. In further embodiments, the side chain of the unnatural amino acid has orthogonal chemistry to that of naturally occurring amino acids. In further embodiments, the side chain of the unnatural amino acid comprises a nucleophile-containing moiety. In further embodiments, the nucleophile-containing moiety in the side chain of the unnatural amino acid can undergo reactions to produce an amine-linked derivatized dolastatin. In further embodiments, the side chain of the unnatural amino acid comprises an electrophile-containing moiety. In further embodiments, the electrophile-containing moiety in the side chain of the unnatural amino acid can undergo nucleophilic attack to generate an amine-linked derivatized dolastatin. In the embodiments described above in this paragraph, the unnatural amino acid can exist as a separate molecule or can be incorporated into a polypeptide of any length. In the latter case, the polypeptide can be naturally occurring or can incorporate additional unnatural amino acids.

[0572] The modification of unnatural amino acids described herein using reductive alkylation or reductive amination reactions has any or all of the following advantages. First, aromatic amines can be reductively alkylated with carbonyl-containing compounds (including aldehydes and ketones) at a pH range of about 4 to about 10 (in some embodiments, a pH range of about 4 to about 7) to generate substituted amines (including secondary and tertiary amines, linkages). Second, because the side chains of naturally occurring amino acids are unreactive under these reaction conditions, the chemistry is selective for unnatural amino acids. This allows for site-specific derivatization of polypeptides incorporating unnatural amino acids (e.g., recombinant proteins) containing aromatic amine or protected aldehyde moieties. This allows for the preparation of such derivatized polypeptides and proteins as defined, homogeneous products. Third, the mild environment required to allow the reaction of an aromatic amine moiety in an amino acid incorporated into a polypeptide with an aldehyde-containing reagent generally does not irreversibly disrupt the tertiary structure of the polypeptide (unless, of course, the purpose of the reaction is to disrupt such tertiary structure). Similarly, the mild environment required to allow the reaction of an aldehyde moiety in an amino acid incorporated into a polypeptide and deprotected with an aromatic amine-containing reagent generally does not irreversibly disrupt the tertiary structure of the polypeptide (unless, of course, the purpose of the reaction is to disrupt such tertiary structure). Fourth, the reaction occurs rapidly at room temperature, thereby allowing the use of many types of polypeptides or reagents that are unstable at high temperatures. Fifth, the reaction occurs readily in aqueous conditions, thereby again allowing the use of polypeptides and reagents that are incompatible (to any degree) with non-aqueous solutions. Sixth, the reaction occurs readily even when the ratio of polypeptide or amino acid to reagent is stoichiometric, approximately stoichiometric, or near-stoichiometric. As a result, it is not necessary to add excess reagents or polypeptides to obtain useful amounts of reaction product.Seventh, the resulting amines can be generated regioselectively and / or regiospecifically by design of the amine and carbonyl moieties in the reactants. Finally, reductive alkylation of aromatic amines with aldehyde-containing reagents and reductive amination of aldehydes with aromatic amine-containing reagents generate amines, including secondary and tertiary amines, and linkages that are stable under biological conditions.

[0573] Unnatural amino acids bearing nucleophilic reactive groups allow for a variety of reactions to link molecules together, including, but not limited to, reductive alkylation reactions using aldehyde-containing dolastatin-linked derivatives. Unnatural amino acids bearing such nucleophilic reactive groups include, by way of example only, aromatic amine groups (including secondary and tertiary amine groups), masked aromatic amine groups (which can be readily converted to aromatic amine groups), or protected aromatic amine groups (which, upon deprotection, have similar reactivity to aromatic amine groups). Such alkylated unnatural amino acid-linked dolastatin derivatives include amino acids having the structure of formula (XXV), (XXXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX).

[0574] [ka] JPEG0007638942000159.jpg220169

[0575] (In the formula, Z has the following structure:

[0576] [ka]

[0577] R5 is H, CO2H, alkyl having 1 to 6 carbon atoms, or thiazole; R6 is OH or H; Ar is phenyl or pyridine; R1 is H, an amino protecting group, a resin, at least one amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, at least one amino acid, a polypeptide, or a polynucleotide; R4 is H, halogen, lower alkyl, or substituted lower alkyl; R7 is alkyl having 1 to 6 carbon atoms or hydrogen; L, L1, L2, L3, and L4 each independently represent a bond, -alkylene-, -alkylene-C(O)-, or -(alkylene-O) n -Alkylene-, -(alkylene-O) n -Alkylene-C(O)-, -(Alkylene-O) n -(CH2) n’ -NHC(O)-(CH2) n’’ -C(Me)2-SS-(CH2) n’’’ -NHC(O)-(alkylene-O) n’’’’ -Alkylene, -(alkylene-O) n -Alkylene-W-, -Alkylene-C(O)-W-, -(Alkylene-O) n -Alkylene-J-, -Alkylene'-J-(Alkylene-O) n -Alkylene-, -(Alkylene-O) n -Alkylene-J-alkylene'-, -J-(alkylene-O) n -Alkylene-, -(Alkylene-O) n -Alkylene-J-(Alkylene-O) n '-Alkylene-J'-, -W-, -Alkylene-W-, Alkylene'-J-(Alkylene-NMe) n -Alkylene-W-, and -J-(Alkylene-NMe) n -Alkylene-W-, -(Alkylene-O) n-alkylene-U-alkylene; -J-alkylene-NMe-alkylene'-NMe-alkylene''-W-, and -alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene'''-W-; W has the following structure:

[0578] [ka]

[0579] U has the structure:

[0580] [ka]

[0581] Each J and J' independently has the structure:

[0582] [ka]

[0583] n and n' are each independently an integer of 1 or greater; Each R 16 are independently selected from the group consisting of hydrogen, halogen, alkyl, NO, CN, and substituted alkyl. Such alkylated non-natural amino acid linked dolastatin derivatives can be in the form of a salt or can be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or polynucleotide and optionally reductively alkylated.

[0584] In some embodiments of a compound of Formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX), R5 is thiazole or carboxylic acid. In some embodiments of a compound of Formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX), R6 is H. In some embodiments of a compound of Formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX), Ar is phenyl. In some embodiments of a compound of Formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX), R7 is methyl. In some embodiments of a compound of Formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX), n is an integer from 0 to 20. In some embodiments of a compound of Formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX), n is an integer from 0 to 10. In some embodiments of a compound of Formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX), n is an integer from 0 to 5.

[0585] In some embodiments of a compound of Formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX), R5 is thiazole or carboxylic acid. In some embodiments of a compound of Formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX), R5 is hydrogen. In some embodiments of a compound of Formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX), R5 is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or hexyl. In some embodiments of a compound of Formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX), R5 is -NH-(alkylene-O) nand the alkylene is -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, or -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-. In certain embodiments of a compound of Formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX), said alkylene is methylene, ethylene, propylene, butylene, pentylene, hexylene, or heptylene.

[0586] In certain embodiments of a compound of Formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX), R5 is -NH-(alkylene-O) n -NH2, and n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

[0587] In some embodiments of a compound of Formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX), R6 is hydrogen. In some embodiments of a compound of Formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX), R6 is hydroxy.

[0588] In certain embodiments of a compound of Formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX), Ar is phenyl.

[0589] In some embodiments of a compound of Formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX), R7 is methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, pentyl, or hexyl. In some embodiments of a compound of Formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX), R7 is hydrogen.

[0590] In some embodiments of a compound of Formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX), L, L1, L2, L3, and L4 are each independently a cleavable linker or a non-cleavable linker. In some embodiments of a compound of Formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX), L, L1, L2, L3, and L4 are each independently an oligo(ethylene glycol)-derivatized linker.

[0591] In certain embodiments of a compound of Formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX), alkylene, alkylene', alkylene'', and alkylene''' are each independently -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, -CH 2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, or -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-. In certain embodiments of a compound of Formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX), alkylene is methylene, ethylene, propylene, butylene, pentylene, hexylene, or heptylene.

[0592] In certain embodiments of a compound of Formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX), n, n', n'', n''', and n''' are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

[0593] In some embodiments of a compound of Formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX), R1 is a polypeptide. In some embodiments of a compound of Formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX), R2 is a polypeptide. In some embodiments of a compound of Formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX), the polypeptide is an antibody. In some embodiments of a compound of Formula (XXV), the antibody is Herceptin.

[0594] Compounds of formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX) are formed by reductive alkylation of aromatic amine compounds with carbonyl containing reagents such as, by way of example, ketones, esters, thioesters, aldehydes, and the like.

[0595] In some embodiments, the masked amine moiety of an unnatural amino acid contained in a polypeptide is first reduced to produce an unnatural amino acid containing an aromatic amine moiety incorporated into the unnatural amino acid polypeptide. This aromatic amine moiety is then reductively alkylated with a carbonyl-containing reagent, as described above, to produce a polypeptide containing an unnatural amino acid of formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX). This reaction also applies to unnatural amino acids incorporated into synthetic polymers, polysaccharides, or polynucleotides. This reaction also applies to unnatural amino acids that are not incorporated. Examples of reducing agents used to reduce the masked amine moiety include, but are not limited to, TCEP, NaS, NaSO, LiAlH, BH, and NaBH. By way of example only, reductive alkylation occurs in an aqueous buffer at a pH of about 4-7 using a mild reducing agent, such as, by way of example only, sodium cyanoborohydride (NaBCNH). Additionally, other reducing agents used in reductive alkylation include, but are not limited to, TCEP, Na2S, Na2S2O4, LiAlH4, B2H6, and NaBH4.

[0596] A non-limiting example of the synthesis of unnatural amino acid polypeptides containing amino acids of formula (XXV), (XXVI), (XXXVII), (XXXVIII), (XXXIX), or (XXX) involves reductive alkylation of the secondary aromatic amine moiety contained in the unnatural amino acid using the carbonyl-containing reagents described above. Such reductive alkylation produces polypeptides containing unnatural amino acids with tertiary aromatic amine moieties. This reaction also applies to unnatural amino acids incorporated into synthetic polymers, polysaccharides, or polynucleotides. This reaction also applies to unincorporated unnatural amino acids. By way of example only, reductive alkylation occurs in aqueous buffer at a pH of about 4-7 using a mild reducing agent, such as, by way of example only, sodium cyanoborohydride (NaBCNH). Additionally, other reducing agents used in reductive alkylation include, but are not limited to, TCEP, NaS, NaSO, LiAlH, BH, and NaBH.

[0597] C. Chemical Synthesis of Unnatural Amino Acid-Linked Dolastatin Derivatives: Heteroaryl-Containing Dolastatin Derivatives In one aspect, the unnatural amino acid is an unnatural amino acid for chemical derivatization of dolastatin-linked derivatives based on the reactivity of dicarbonyl groups, including groups containing at least one ketone group, at least one aldehyde group, at least one ester group, at least one carboxylic acid, and / or at least one thioester group, where the dicarbonyl group can be a 1,2-dicarbonyl group, a 1,3-dicarbonyl group, or a 1,4-dicarbonyl group. In further or additional aspects, the unnatural amino acid is an unnatural amino acid for chemical derivatization of dolastatin-linked derivatives based on the reactivity of diamine groups, including hydrazine groups, amidine groups, imine groups, 1,1-diamine groups, 1,2-diamine groups, 1,3-diamine groups, and 1,4-diamine groups. In further or additional embodiments, at least one of the above unnatural amino acids is incorporated into the dolastatin-linked derivative. Thus, such embodiments are non-natural amino acid-linked dolastatin derivatives. In further or additional embodiments, the non-natural amino acid is functionalized at its side chain such that reaction of the non-natural amino acid with a derivatizing molecule generates a linkage that includes a heterocycle-based linkage and / or an aldol-based linkage, including a nitrogen-containing heterocycle. In further or additional embodiments, the non-natural amino acid is a non-natural amino acid polypeptide that can react with a derivatized dolastatin linker to generate a linkage that includes a heterocycle-based linkage and / or an aldol-based linkage, including a nitrogen-containing heterocycle. In further or additional embodiments, the non-natural amino acid is selected from amino acids having dicarbonyl and / or side chain diamines. In further or additional embodiments, the non-natural amino acid includes a masked side chain group, including a masked diamine group and / or a masked dicarbonyl group.In further or additional embodiments, the unnatural amino acid comprises a group selected from a ketoamine (i.e., a group that contains both a ketone and an amine), a ketoalkyne (i.e., a group that contains both a ketone and an alkyne), and an enedione (i.e., a group that contains both a dicarbonyl group and an alkene).

[0598] In further or additional embodiments, the unnatural amino acid comprises a dicarbonyl side chain, where the carbonyl is selected from a ketone, an aldehyde, a carboxylic acid, or an ester, including a thioester. In another embodiment, the unnatural amino acid is an unnatural amino acid that comprises a functional group that can form a heterocycle, including a nitrogen-containing heterocycle, upon appropriate treatment with a functionalizing reagent. In further or additional embodiments, the unnatural amino acid resembles a natural amino acid in structure, but includes any one of the above functional groups. In another or additional embodiment, the unnatural amino acid resembles phenylalanine or tyrosine (aromatic amino acids), while in another embodiment, the unnatural amino acid resembles alanine and leucine (...

Claims

1. A compound having the following structure, or a salt thereof: 【Chemistry 1】

2. A compound having the following structure, or a salt thereof: 【Chemistry 2】

3. A compound having the following structure, or a salt thereof: 【Chemistry 3】

4. A compound having the following structure, or a salt thereof: 【Chemistry 4】

5. A compound according to any one of claims 1 to 4, A pharmaceutical composition comprising a pharma- ceutically acceptable carrier, excipient, or binder.

6. 13. Use of a compound of claim 1 as an intermediate in a process for the preparation of a derivatized dolastatin analogue, comprising: The derivatized dolastatin analog has at least one oxime-containing amino acid, The process comprises the step of contacting the compound with a reagent of formula (XXXVII), The dolastatin analogue has the formula (VIIIa): 【Chemistry 5】 During the ceremony, A is optionally lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, when present; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) ｋ - (where k is 1, 2, or 3), -S(O) ｋ (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) ｋ N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) ｋ N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R') ２ -N=N- and -C(R') ２ is a linker selected from the group consisting of -N(R')-N(R')-; each R' is independently H, alkyl, or substituted alkyl; K. 【Chemistry 6】 and R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R １ is H, an amino protecting group, a resin, at least one amino acid, or a polynucleotide; R ２ is OH, an ester protecting group, a resin, at least one amino acid, or a polynucleotide; R ３ and R ４ are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R ３ and R ４ , or two R ３ The group may optionally form a cycloalkyl or heterocycloalkyl. 【Chemistry 7】 7. Use of a compound according to claim 2 as an intermediate in a process for the preparation of a derivatized dolastatin analogue, comprising the steps of: The derivatized dolastatin analog has at least one oxime-containing amino acid, The process comprises the step of contacting the compound with a reagent of formula (XXXVII), The dolastatin analogue is represented by formula (VIIIb): 【Chemistry 8】 During the ceremony, A is optionally lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, when present; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (wherein k is 1, 2, or 3), -S(O) k . (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O)k a linker selected from the group consisting of N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R') 2 -N=N-, and -C(R') 2 -N(R')-N(R')-; each R' is independently H, alkyl, or substituted alkyl; K. 【Chemistry 9】 and R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R 1 is H, an amino protecting group, a resin, at least one amino acid, or a polynucleotide; R 2 is OH, an ester protecting group, a resin, at least one amino acid, or a polynucleotide; R 3 and R 4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R 3 and R 4 or two R 3 groups may optionally form a cycloalkyl or heterocycloalkyl. 【Chemistry 10】 8. Use of a compound according to claim 3 as an intermediate in a process for the preparation of a derivatized dolastatin analogue, comprising the steps of: The derivatized dolastatin analog has at least one oxime-containing amino acid, The process comprises the step of contacting the compound with a reagent of formula (XXXVII), The dolastatin analogue has the formula (VIIIc): 【Chemistry 11】 During the ceremony, A is optionally lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, when present; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (wherein k is 1, 2, or 3), -S(O) k . (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O)k a linker selected from the group consisting of N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R') 2 -N=N-, and -C(R') 2 -N(R')-N(R')-; each R' is independently H, alkyl, or substituted alkyl; K. 【Chemistry 12】 and R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R 1 is H, an amino protecting group, a resin, at least one amino acid, or a polynucleotide; R 2 is OH, an ester protecting group, a resin, at least one amino acid, or a polynucleotide; R 3 and R 4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R 3 and R 4 or two R 3 groups may optionally form a cycloalkyl or heterocycloalkyl. 【Chemistry 13】 9. Use of a compound according to claim 4 as an intermediate in a process for the preparation of a derivatized dolastatin analogue, comprising the steps of: The derivatized dolastatin analog has at least one oxime-containing amino acid, The process comprises the step of contacting the compound with a reagent of formula (XXXVII), The dolastatin analogue is represented by formula (VIIId): 【Chemistry 14】 During the ceremony, A is optionally lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, when present; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (wherein k is 1, 2, or 3), -S(O) k . (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O)k a linker selected from the group consisting of N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R') 2 -N=N-, and -C(R') 2 -N(R')-N(R')-; each R' is independently H, alkyl, or substituted alkyl; K. 【Chemistry 15】 and R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R 1 is H, an amino protecting group, a resin, at least one amino acid, or a polynucleotide; R 2 is OH, an ester protecting group, a resin, at least one amino acid, or a polynucleotide; R 3 and R 4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R 3 and R 4 or two R 3 groups may optionally form a cycloalkyl or heterocycloalkyl. 【Chemistry 16】

10. 2. Use of a compound according to claim 1 in the manufacture of a medicament for treating a disease, comprising The preparation includes the step of derivatizing the compound to form a dolastatin analog having at least one oxime-containing amino acid; The derivatization comprises contacting the compound with a reagent of formula (XXXVII), The dolastatin analogue has the formula (VIIIa): 【Chemistry 17】 During the ceremony, A is optionally lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, when present; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) ｋ - (where k is 1, 2, or 3), -S(O) ｋ (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) ｋ N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) ｋ N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R') ２ -N=N- and -C(R') ２ is a linker selected from the group consisting of -N(R')-N(R')-; each R' is independently H, alkyl, or substituted alkyl; K. 【Chemistry 18】 and R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R １ is H, an amino protecting group, a resin, at least one amino acid, or a polynucleotide; R ２ is OH, an ester protecting group, a resin, at least one amino acid, or a polynucleotide; R ３ and R ４ are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R ３ and R ４ , or two R ３ The group may optionally form a cycloalkyl or heterocycloalkyl. 【Chemistry 19】 11. Use of a compound according to claim 2 in the manufacture of a medicament for treating a disease, comprising: The preparation includes the step of derivatizing the compound to form a dolastatin analog having at least one oxime-containing amino acid; The derivatization comprises contacting the compound with a reagent of formula (XXXVII), The dolastatin analogue is represented by formula (VIIIb): 【Chemistry 20】 During the ceremony, A is optionally lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, when present; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (wherein k is 1, 2, or 3), -S(O) k . (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O)k a linker selected from the group consisting of N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R') 2 -N=N-, and -C(R') 2 -N(R')-N(R')-; each R' is independently H, alkyl, or substituted alkyl; K. 【Chemistry 21】 and R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R 1 is H, an amino protecting group, a resin, at least one amino acid, or a polynucleotide; R 2 is OH, an ester protecting group, a resin, at least one amino acid, or a polynucleotide; R 3 and R 4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R 3 and R 4 or two R 3 groups may optionally form a cycloalkyl or heterocycloalkyl. 【Chemical 22】 12. Use of a compound according to claim 3 in the manufacture of a medicament for treating a disease, comprising: The preparation includes the step of derivatizing the compound to form a dolastatin analog having at least one oxime-containing amino acid; The derivatization comprises contacting the compound with a reagent of formula (XXXVII), The dolastatin analogue has the formula (VIIIc): 【Chemistry 23】 During the ceremony, A is optionally lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, when present; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (wherein k is 1, 2, or 3), -S(O) k . (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O)k a linker selected from the group consisting of N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R') 2 -N=N-, and -C(R') 2 -N(R')-N(R')-; each R' is independently H, alkyl, or substituted alkyl; K. 【Chemistry 24】 and R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R 1 is H, an amino protecting group, a resin, at least one amino acid, or a polynucleotide; R 2 is OH, an ester protecting group, a resin, at least one amino acid, or a polynucleotide; R 3 and R 4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R 3 and R 4 or two R 3 groups may optionally form a cycloalkyl or heterocycloalkyl. 【Chemistry 25】 13. Use of a compound according to claim 4 in the manufacture of a medicament for treating a disease, comprising: The preparation includes the step of derivatizing the compound to form a dolastatin analog having at least one oxime-containing amino acid; The derivatization comprises contacting the compound with a reagent of formula (XXXVII), The dolastatin analogue is represented by formula (VIIId): 【Chemistry 26】 During the ceremony, A is optionally lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, when present; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (wherein k is 1, 2, or 3), -S(O) k . (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O)k a linker selected from the group consisting of N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R') 2 -N=N-, and -C(R') 2 -N(R')-N(R')-; each R' is independently H, alkyl, or substituted alkyl; K. 【Chemical 27】 and R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R 1 is H, an amino protecting group, a resin, at least one amino acid, or a polynucleotide; R 2 is OH, an ester protecting group, a resin, at least one amino acid, or a polynucleotide; R 3 and R 4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R 3 and R 4 or two R 3 groups may optionally form a cycloalkyl or heterocycloalkyl. 【Chemistry 28】

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